Derivatives of amanita toxins and their conjugation to a cell binding molecule

ABSTRACT

The present invention is related to novel cytotoxic agents, derivatives of  Amanita  toxins of Formula (I), wherein  , -----, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , X, L, m, n and Q are defined herein, the preparation and the therapeutic uses in the targeted treatment of cancers, autoimmune disorders, and infectious diseases.

FIELD OF THE INVENTION

The present invention relates to novel cytotoxic agents, derivatives ofAmanita toxins and their uses to specifically target cell population bychemically linking these derivatives to a cell binding agent.

BACKGROUND OF THE INVENTION

Antibody-drug conjugates (ADCs), which utilize the targeting ability ofmonoclonal antibodies (mAbs) to deliver highly toxic drugs to targetedtumor cells, have been proof that the products are technically feasible,therapeutically effective, and able to get regulatory approval afterSeattle Genetics/Takeda's Brentuximab vedotin (Adcetris) andImmunoGen/Roche's Trastuzumab emtansine (Kadcyla) are on the market(Sassoon, I. and Blanc, V. Laurent Ducry (ed.), Antibody-DrugConjugates, Methods in Molecular Biology, vol. 1045, p 1-27). Currentlythere are more than 50 ADC drugs in the clinic trials according towww.clinictrails.gov and many big pharmas and biotech firms, as well asplenty of small start-ups have a lot of incentives to invest in furtherdevelopment of this technology. Indeed, the complexity of antibody-drugconjugates with its four moving repertoires of cytotoxic agents, linkertechnologies, antibody properties, and conjugation methods makes thisfield incredibly challenging yet full of potential for innovation(Perez, H. L., et al. Drug Discovery Today, 2014, 19(7), 869-81). Thedevelopment from novel targeting ligands to new site specificconjugation methods, from multiple payloads to changing thedrug-antibody ratio, from functional linkers to homogeneousstoichiometry, are challenging the convention for the design anddevelopment of next-generation ADCs (Zhao, R. et al, PCT/182015/055051,PCT/IB2015/055264, introduction part). However, the important key factorin generating powerful ADCs is the cytotoxic agents (Bourchard, H., etal, Bioorg. Med. Chem. Lett. 2014, 24, 5357-63). One class of the highlypotent cytotoxic agents that can be used for ADC is the Amanitins(Moldenhauer, G. et al, J Natl Cancer Inst 2012, 104 (8): 622-34;Flygare, J. A. et al, Chem. Biol. Drug. Des. 2013, 81, 113-121;WO2010/115629, WO2010/115630, WO2012041504, WO2012119787, WO2014/009025,WO2014135282, US pat appl 20120213805, 20120100161, 20130259880,20140294865, 20150218220, EP2416805, EP1661584, EP1859811, EP2497499,EP2774624), which are highly toxic components of Amanita toxins (Vetter,J., Toxicon 1998, 36 (I): 13-24).

Amanita toxins are mainly cyclic octa- and heptapeptide toxins ofAmanita mushrooms, which contain an unusual Trp-Cys crossbridge known astryptathionine (Walton, J. D. et al. Biopolymers 2010; 94(5): 659-64).The major components of amanita toxins are amatoxins, phallotoxins, andvirotoxins (Wieland, T., Faulstich, H., CRC Crit. Rev. Biochem. 1978,5(3):185-260; Vetter, J., Toxicon 1998, 36 (1): 13-24; Weiland, T., andFaulstich, H. 1983. Peptide Toxins from Amanita. p. 585-635. In:Handbook of Natural Toxins, Volume I: Plant and Fungal Toxins. R. F.Keeler and A. T. Tu, Ed. Marcel Dekker, Inc. New York, N.Y., Wieland,T., Int J. Pept. Protein Res., 1983, 22, 257-76) and their collectivelyknown structures are listed in Tables 1, 2 and 3 respectively below.

TABLE 1 Ten currently known structures of amatoxins

Name R₁ R₂ R₃ R₄ R₅ α-Amanitin OH OH NH₂ OH OH β-Amanitin OH OH OH OH OHγ-Amanitin H OH NH₂ OH OH ε-Amanitin H OH OH OH OH Amanullin H H NH₂ OHOH Amanullinic acid H H OH OH OH Amaninamide OH OH NH₂ H OH Amanin OH OHOH H OH Proamanullin H H H NH₂ OH H

(The δ-Amanitin has been reported, but its chemical structure has notbeen determined)

TABLE 2 Structures of the Phallotoxins

Name R₁′ R₂′ R₃′ R₄′ R₅′ phalloidin OH H CH₃ CH₃ OH phalloin H H CH₃ CH₃OH prophallin H H CH₃ CH₃ H phallisin OH OH CH₃ CH₃ OH phallacin H HCH(CH₃)₂ COOH OH phallacidin OH H CH(CH₃)₂ COOH OH phallisacin OH OHCH(CH₃)₂ COOH OH

TABLE 3 Structures of the Virotoxins

Name R_(l)″ R₂″ X Viroidin CH₃ CH(CH₃)₂ S(O₂) Desoxiviroidin CH₃CH(CH₃)₂ S(O) (Ala)viroidin CH₃ CH₃ S(O₂) (Ala)desoxiviroidin CH₃ CH₃S(O) (Ala)viroisin CH₂OH CH(CH₃)₂ S(O₂) Desoxiviroisin CH₂OH CH(CH₃)₂S(O)

Amatoxins which are a subgroup of at least ten toxic compoundsoriginally found in several genera of poisonous mushrooms, most notablyAmanita phalloides and several other mushroom species, are bicyclicoctapeptides containing intra-annular tryptathionine crossbridge that isoxidatively formed from tryptophan and cysteine (Kaya, E., et al,Toxicon 2013, 76, 225-33).

Amatoxins are potent and selective inhibitors of RNA polymerase II (PolII), a vital enzyme in the synthesis of messenger RNA (mRNA), microRNA,and small nuclear RNA (snRNA) (Karlson-Stiber C, Persson H. 2003“Cytotoxic fungi—an overview”, Toxicon 42 (4): 339-49). Thus Amatoxinskill cells by shutting down gene transcription and protein biosynthesis(Brodner, 0. G. and Wieland, T. 1976 Biochem., 15(16): 3480-4; Fiume,L., Curr Probl Clin Biochem, 1977, 7: 23-8; Karlson-Stiber C, Persson H.2003, Toxicon 42(4): 339-49; Chafin, D. R., Guo, H. & Price, D. H. 1995J. Biol. Chem. 270 (32): 19114-19; Wieland (1983) Int. J. Pept. ProteinRes. 22(3): 257-76). So far ten known amatoxins, named α-Amanitin,β-Amanitin, γ-Amanitin, ε-Amanitin, Amanullin, Amanullinic acid,Amaninamide, Amanin, and Proamanullin are synthesized as 35-amino-acidproproteins, from which the final eight amino acids are cleaved by aprolyl oligopeptidase (Litten, W. 1975 Scientific American 232 (3):90-101; H. E. Hallen, et al 2007 Proc. Nat. Aca. Sci. USA 104,19097-101; K. Baumann, et al, 1993 Biochemistry 32 (15): 4043-50;Karlson-Stiber C, Persson H. 2003, Toxicon 42 (4): 339-49; Horgen, P. A.et al. 1978 Arch. Microbio. 118 (3): 317-9). Amatoxins can be producedfrom collected Amanita phalloides mushrooms (Yocum, R. R. 1978Biochemistry 17(18): 3786-9; Zhang, P. et al, 2005, FEMS Microbiol.Lett. 252(2), 223-8), or from fermentation using a basidiomycete(Muraoka, S. and Shinozawa T., 2000 J. Biosci. Bioeng. 89(1): 73-6, USpat. Appl 20100267019) or from fermentation using A. fissa (Guo, X. W.,et al, 2006 Wei Sheng Wu Xue Bao 46(3): 373-8), or from culturingGalerina fasciculata or Galerina helvoliceps (WO/1990/009799,JP11137291). However the yields from these isolation and fermentationwere quite low (less than 5 mg/L culture). Several semi-chemical orsynthetic preparations of amatoxins and their analogs have been reportedin the past three decades (W. E. Savige, A. Fontana, Chem. Commun. 1976,600-1; Zanotti, G., et al, Int J Pept Protein Res, 1981. 18(2): 162-8;Wieland, T., et al, Eur. J. Biochem. 1981, 117, 161-4; P. A. Bartlett,et al, Tetrahedron Lett. 1982, 23, 619-22; Zanotti, G., et al., BiochimBiophys Acta, 1986. 870 (3): 454-62; Zanotti, G., et al., Int. J.Peptide Protein Res. 1987, 30, 323-9; Zanotti, G., et al., Int. J.Peptide Protein Res. 1987, 30, 450-9; Zanotti, G., et al., Int J PeptProtein Res, 1988. 32(1): 9-20; G. Zanotti, T. et al, Int. J. PeptideProtein Res. 1989, 34, 222-8; Zanotti, G., et al., Int J Pept ProteinRes, 1990. 35(3): 263-70; Mullersman, J. E. and J. F. Preston, 3rd, IntJ Pept Protein Res, 1991. 37(6): 544-51; Mullersman, J. E., et al, Int JPept Protein Res, 1991. 38(5): 409-16; Zanotti, G., et al, Int J PeptProtein Res, 1992. 40(6): 551-8; Schmitt, W. et al, J. Am, Chem. Soc.1996, 118, 4380-7; Anderson, M. O., et al, J. Org. Chem., 2005, 70(12):4578-84; J. P. May, et al, J. Org. Chem. 2005, 70, 8424-30; F.Brueckner, P. Cramer, Nat. Struct. Mol. Biol. 2008, 15, 811-8; J. P.May, D. M. Perrin, Chem. Eur. J. 2008, 14, 3404-9; J. P. May, et al,Chem. Eur. J. 2008, 14, 3410-17; Q. Wang, et al, Eur. J. Org. Chem.2002, 834-9; May, J. P. and D. M. Perrin, Biopolymers, 2007. 88(5):714-24; May, J. P., et al., Chemistry, 2008. 14(11): 3410-7; S. De LamoMarin, et al, Eur. J. Org. Chem. 2010, 3985-9; Pousse, G., et al., OrgLett, 2010. 12(16): 3582-5; Luo, H., et al., Chem Biol, 2014. 21(12):1610-7; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5). Because oftheir extreme potency and unique mechanism of cytotoxicity, amatoxinshave been used as payloads for conjugations (Fiume, L., Lancet, 1969. 2(7625): 853-4; Barbanti-Brodano, G. and L. Fiume, Nat New Biol, 1973.243(130): 281-3; Bonetti, E., M. et al, Arch Toxicol, 1976. 35(1): p.69-73; Davis, M. T., Preston, J. F. Science 1981, 213, 1385-1388;Preston, J. F., et al, Arch Biochem Biophys, 1981. 209(1): 63-71; H.Faulstich, et al, Biochemistry 1981, 20, 6498-504; Barak, L. S., et al.,Proc Natl Acad Sci USA, 1981. 78(5): 3034-8; Faulstich, H. and L. Fiume,Methods Enzymol, 1985. 112: 225-37; Zhelev, Z., A. et al, Toxicon, 1987.25(9): 981-7; Khalacheva, K., et al, Eksp Med Morfol, 1990. 29(3):26-30; U. Bermbach, H. Faulstich, Biochemistry 1990, 29, 6839-45;Mullersman, J. E. and J. F. Preston, Int. J. Peptide Protein Res. 1991,37, 544-51; Mullersman, J. E. and J. F. Preston, Biochem Cell Biol,1991. 69(7): 418-27; J. Anderl, H. Echner, H. Faulstich, Beilstein J.Org. Chem. 2012, 8, 2072-84; Moldenhauer, G., et al, J. Natl. CancerInst. 2012, 104, 622-34; A. Moshnikova, et al; Biochemistry 2013, 52,1171-8; Zhao, L., et al., Chembiochem, 2015. 16(10): 1420-5; Zhou, B.,et al., Biosens Bioelectron, 2015. 68: 189-96; WO2014/043403,US20150218220, EP 1661584).

The phallotoxins which are bicyclic heptapeptides, consist of at leastseven compounds as shown in Table 2, were originally discovered from thedeath cap mushroom (Amanita phalloides) in 1937 by Feodor Lynen,Heinrich Wieland's student and son-in-law, and Ulrich Wieland of theUniversity of Munich (Enjalbert, F., et al, Toxicon 1993, 31, 803-7).The phallotoxins, after i.p. administration, inhibit the conversion ofactin-F into actin-G and disturb the dynamic equilibrium of these formsnecessary for cell functions (Enjalbert, F., et al., C. R. Acad. Sci.Paris, Sciences de la vie/Life Sciences, 1999, 322, 855-62; Wieland, T.,50 Jahre Phalloidin, Naturwissenchaften 1987, 74, 367-73). There are sixknown structures of phallotoxins namely prophalloin, phalloin,phallisin, phallacidin, phallacin and phallisacin as shown in Table 2(Yocum R. R., Simons M., Lloydia 1977, 40, 178-90; Enjalbert, F., etal., C. R. Acad. Sci. Paris, Sciences de la vie/Life Sciences, 1999,322, 855-62; Schafer A. J., Faulstich H., Anal. Biochem. 1977, 83,720-723). Though highly toxic to liver cells, phallotoxins have sincebeen found to have little contribution to the death cap's toxicitybecause they are not absorbed through the gut. On average, the sixstructure known phallotoxins are about 5-10 times less toxic than (α-,β-, γ-, or ε-) amanitins (Vetter, J., Toxicon, 1998, 36, 13-24).Furthermore, phalloidin is also found in the edible Blusher (Amanitarubescens) (Litten, W., Scientific American 1975, 232, 90-101).

Virotoxins are monocyclic heptapeptides formed by at least fivedifferent compounds: alaviroidin, viroisin, deoxoviroisin, viroidin, anddeoxoviroidin (Vetter, J., Toxicon, 1998, 36, 13-24) as shown in Table3. The structure and biological activity of virotoxins are similar tothose of phallotoxins, thus suggesting that virotoxins arebiosynthetically derived from phallotoxins or share common precursorpathways (Wieland, T., Int J Pept Protein Res, 1983, 22, 257-76).However, differing from phallotoxins, virotoxins are monocyclic peptidesand contain D-serine instead of L-cysteine, as well as have twounnatural amino acids: 2,3-trans-3,4-dihydroxy-L-proline and2′-(methylsulfonyl)-L-tryptophan (Buku, A. et al, Proc Natl Acad SciUSA. 1980, 77(5), 2370-1). NMR studies revealed that D-configuration ofserine is the structural element that maintains the phalloidin-likestructure, while the hydroxy group does not contribute to conformationalstability but is likely to be in contact with the actin surface(Zanotti, G., et al, Biochemistry. 1999, 38(33):10723-9). On themolecular level, the virotoxins behave similarly to the phallotoxins,e.g. the affinity of viroisin is very similar to that of phalloidin,with an apparent equilibrium dissociation constant K_(d) approximately2×10⁻⁸M (Faulstich, H., et al, Biochemistry, 1980, 19, 3334-43).However, the flexibility of the monocyclic structure and the presence oftwo additional hydroxy groups in the virotoxins suggest a different modeof interaction with actin. While there is proof that the bicyclicphallotoxins possess a rigid binding site, the virotoxins may adopt thebiologically active conformation by an induced-fit mechanism uponcontacting with actin (Faulstich, H., et al, Biochemistry, 1980, 19,3334-43).

There are several methods disclosed so far in conjugation of amatoxins.Preston and his collegues used diazotization ofp-aminobenzoylglycylglycine and then coupling the linker to a-amanitinat the 7′C position of Trptophan (Preston, J. F. et al, Arch. Biochem.Biophy. 1981, 209, 63-71; Davis, M-T. B. and Preston, J. F., Science,1981, 213, 1385-7; Hencin, R. S. and Preston, J. F., Mol. Pharm., 1979,16, 961-9). Heidelberg Pharma GMBH (WO2010/115629, WO2010115630,WO2012/041504, US20120100161, US20120213805) disclosed the conjugationof amatoxin via the oxygen atom as an ether linkage to the 6′C-atom ofamino acid 4, or via the oxygen atom as an ester or a carbomate linkageto the SC-atom of amino acid 3, or via the nitrogen atom as an amidelinkage to the γC-atom of amino acid 1. Heidelberg Pharma GMBH(EP2774624, WO2012119787, WO2014/135282, US20140294865, US20160002298)also disclosed the conjugation via position of 1′N-atom of indole ofamatoxin. Agensys Inc (US20150218220) disclosed that through activationof the C7′-position of the indole group of a-amanitin with a reagentsuch as iodine, followed by coupling with a suitably substituted2°-amino reagent to introduce a diamine spacer linkage, or introduce acarbon spacer (C—C bond of the C7-position of the indole) in front of a2°-amino linkage in the presence of formaldehyde.

It is known that amatoxins are relatively non-toxic when coupled tolarge biomolecule carriers, such as antibody molecules, and that theyexert their cytotoxic activity only after the biomolecule carrier iscleaved off. In light of the toxicity of amatoxins, particularly theirnotorious toxicity for liver cells (Zhou, P. et al, World J.Gastroenterology, 2012, 18(5), 435-4; Giannini, A., et al, Clin.Toxicology, 2007. 45(5), 539-42), it is of outmost importance thatamatoxin conjugates for targeted tumour therapy remain highly stableafter administration in plasma, and that the released the amatoxin wouldnot run away from the target cells or damage liver cells when occursafter internalization in the target cells.

Here we disclose novel amanita toxin derivatives that first can beconjugated cell-binding molecule through stable amide bonds after simplenitrition, following by reduction and conjugation at the indole unit ofthe amanita toxin derivatives. Second, many of the amanita toxinderivatives of this invention have prodrug units on the indole unitswhich can bring down the highly potency in vitro, but can be slowlytransformed back the highly potent cytotoxicity in vivo after theirextra prodrug units are removed by enzymes. Since amatoxins arenotorious for their extremely toxic to hepatitis with centrolobularnecrosis and hepatic steatosis, as well as acute tubulointerstitialnephropathy, which altogether induce a severe hepatorenal syndrome(Litten, W. 1975 Scientific American 232: 90-10; Karlson-Stiber C,Persson H. 2003 Toxicon 42 (4): 339-49). The slow conversion of thesafer prodrugs to their mother toxins would minimize the severe sidetoxicity when the toxins are off-targets during the delivery. Thus theseimprovements of the conjugatable amanita toxin derivatives may havedrastic consequences of wider therapeutic index windows and much moresafety of the amatoxin conjugates for targeted therapeutic applications.

SUMMARY OF THE INVENTION

The first embodiment of this invention is to disclose potent cytotoxicagents, specifically, derivatives of amatoxins, phallotoxins orvirotoxins which can be effectively used to block cell proliferation. Inparticular, this invention is to disclose novel amanita toxinderivatives, optionally linkable or linked to a cell binding agent toblock cell proliferation. The novel cytotoxic agents and theirconjugates to a cell binding agent of this invention are illustrated inthe following formula (I):

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers.

Wherein

---- represents a single bond that links any carbon position of thearomatic (indole) ring;

represents an optional single bond or an absent bond.

R₁ and R₂ are independently selected from H, OH, CH₂OH, CH(OH)CH₂OH,CH(CH₃)CH₂OH, CH(OH)CH₃, C₁-C₈ alkyl, —OR₁₂ (ether), C₂-C₈ alkenyl,alkynyl, heteroalkyl, —OCOR₁₂ (ester), —OC(═O)OR₁₂-(carbonate),—OC(═O)NHR₁₂ (carbamate); C₃-C₈ aryl, heterocyclic, carbocyclic,cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl.

R₃ and R₄ are independently selected from H, OH, —OR₁₂ (ether), —OCOR₁₂(ester), —OCOCH₃ (acetate), —OCOOR₁₂ (carbonate), —OC(═O)NHR₁₂(carbamate), —OP(O)(OR₁₂)(OR₁₂′) (phosphate), OP(O)(NHR₁₂)(NHR₁₂′)(phosphamide), O—SO₃ ⁻, or O-glycoside;

R₅ is selected from H, OH, NH₂, NHOH, NHNH₂, —OR₁₂, —NHR₁₂, NHNHR₁₂,—NR₁₂R₁₂′, N(H)(R₁₂)R₁₃CO(Aa)_(p), (an amino acid or peptide, wherein Aais an amino acid or a polypeptide, p represents 0-6);

R₆ is selected from H, OH, CH₂OH, CH(OH)CH₂OH, CH(CH₂OH)₂, CH(CH₃)OH,CH₂CH₂OH, PrOH, BuOH, C₁-C₈ alkyl, —OR₁₂ (ether), C₂-C₈ alkenyl,alkynyl, heteroalkyl, —OCOR₁₂ (ester); C₃-C₈ aryl, heterocyclic, orcarbocyclic.

R₇, R₈ and R₉ are independently selected from H, OH, CH₃, CH(CH₃)₂,CH(CH₃)CH₂CH₃, CH₂OH, CH(OH)CH₂OH, CH₂CH(OH)CH₂OH, CH(CH₂OH)₂,CH₂C(OH)(CH₂OH)₂, CH₂C(OH)(CH₃)(CH₂OH), CH₂C(OH)(CH(CH₃)₂)(CH₂OH),CH₂CH₂OH, PrOH, BuOH, CH₂COOH, CH₂CH₂COOH, CH(OH)COOH, CH₂CONH₂,CH₂CH₂CONH₂, CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHC(═NH)NH₂, C₁˜C₈ alkyl, CH₂Ar,CH₂SH, CH₂SR₁₂, CH₂SSR₁₂, CH₂SSAr, CH₂CH₂SCH₃, —OR₁₂ (ether), C₂˜C₈alkenyl, alkynyl, heteroalkyl, —OCOR₁₂ (ester); C₃˜C₈ aryl,heterocyclic, or carbocyclic.

R₁₀ is selected from H, NH₂, OH, SH, NO₂, halogen, —NHOH, —N₃ (azido);—CN (cyano); C₁˜C₈ alkyl, C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈aryl, heterocyclic, or carbocyclic; —OR₁₂ (ether), —OCOR₁₂ (ester),—OCOCH₃ (acetate), —OC(O)OR₁₂ (carbonate), —OC(O)CH(R₁₂)NHAa (Aa is anamino acid group), —NR₁₂R₁₂′(amine), —NR₁₂COR₁₂′(amine), —NR₁₂NR₁₂′NR₁₂″(amine); —OCONR₁₂R₁₂′(carbamate); —NR₁₂(C═NH)NR₁₂′R₁₂″ (guanidinum);—NR₁₂CO(Aa)_(p), (an amino acid or peptide, wherein Aa is an amino acidor a polypeptide, p represents 0-6); —N(R₁₂)CONR₁₂′R₁₂″ (urea);—OCSNHR₁₂ (thiocarbamate); —SH (thiol); —SR₁₂ (sulfide); —S(O)R₁₂(sulfoxide); —S(O₂)R₁₂ (sulfone); —SO₃, HSO₃, HSO₂, or a salt of HSO₃ ⁻,SO₃ ²⁻ or —HSO₂ ⁻ (sulphite); —OSO₃ ⁻; —N(R₁₂)SOOR₁₂′ (sulfonamide);H₂S₂O₅ or a salt of S₂O₅ ²⁻ (metabisulfite); PO₃SH₃, PO₂S₂H₂, POS₃H₂,PS₄H₂ or a salt of PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ³⁻, PS₄ ³⁻ (mono-, di-, tri-,and tetra-thiophosphate); (R₁₂O)₂POSR₁₂′ (thiophosphate ester); HS₂O₃ ora salt of S₂O₃ ²⁻ (thiosulfate); HS₂O₄ or a salt of S₂O₄ ²⁻(dithionite); (P(═S)(OR₁₂)(S)(OH) or a salt formed with a cation(phosphorodithioate); —N(R₁₂)OR₁₂′ (hydroxylamine derivative);R₁₂C(═O)NOH or a salt formed with a cation (hydroxamic acid); (HOCH₂SO₂⁻, or its salts (formaldehyde sulfoxylate); —N(R₁₂)COR₁₂′ (amide);R₁₂R₁₂′R₁₂″NPO₃H (trialkylphosphoramidate or phosphoramidic acid); orArAr′Ar″NPO₃H (triarylphosphonium); OP(O)(OM₁)(OM₂),OCH₂OP(O)(OM₁)(OM₂), OSO₃M₁; O-glycoside (glucoside, galactoside,mannoside, glucuronoside, alloside, fructoside, etc), NH-glycoside,S-glycoside or CH₂-glycoside; M₁ and M₂ are independently H, Na, K, Ca,Mg, NH₄, NR₁′R₂′R₃′; R₁′, R₂′ and R_(3′) are independently H, C₁˜C₈alkyl; Ar, Ar′, and Ar″ are C₃-C₈ aryl or heteroaromatic group.

R₁₁ is H, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl,heteraoaryl.

R₁₂, R₁₂′, and R₁₂″ are independently selected from H, C₁˜C₈ alkyl;C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl, heteraoaryl,heterocyclic, or carbocyclic.

X is S, O, NH, SO, SO₂, or CH₂.

m is 0 or 1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20.

L is a linker or a linker-cell-binding molecule (Q) covalently boundcluster, or a linker which has a functional group on the linker thatenables linkage with a cell-binding agent (CBA). L is preferably areleasable linker, which has the formula of: -Ww-(Aa)r-Tt-; or-Ww-(Aa)r-Tt-Q; or Q-Ww-(Aa)r-Tt-; wherein W is a Stretcher unit; w is 0or 1; Aa is an Amino Acid unit comprising independent amino acids; r isan integer ranging from 0 to 100. The Stretcher unit W may contain aself-immolative or a non-self-immolative component, peptidyl units, ahydrazone bond, a disulfide, an ester, an oxime, an amide, or athioether bond. The self-immolative unit includes, but is not limitedto, aromatic compounds that are electronically similar to thepara-aminobenzylcarbamoyl (PAB) groups such as2-aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs,beta-glucuronide, and ortho or para-aminobenzylacetals. Preferably, theself-immolative linker component has one of the following structures:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker units, or the cytotoxic agent, and/or the bindingmolecule (CBA); X¹, Y¹, Z² and Z³ are independently NH, O, or S; Z¹ isindependently H, NH, O or S; v is 0 or 1; Q¹ is independently H, OH,C₁˜C₆ alkyl, (OCH₂CH₂)_(n), F, Cl, Br, I, OR₁₂, SR₁₂, NR₁₂R₁₂′, N═NR₁₂,N═R₁₂, NR₁₂R₁₂′, NO₂, SOR₁₂R₁₂′, SO₂R₁₂, SO₃R₁₂, OSO₃R₁₂, PR₁₂R₁₂′,POR₁₂R₁₂′, PO₂R₁₂R₁₂′, OPO(OR₁₂)(OR₁₂′), or OCH₂PO(OR₁₂(OR₁₂′) whereinR₁₂ and R₁₂′ are as defined above; preferably R₁₂ and R₁₂′ areindependently selected from H, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl,heteroalkyl; C₃˜C₈ aryl, heterocyclic, carbocyclic, cycloalkyl,heterocycloalkyl, heteroaralkyl, alkylcarbonyl; or pharmaceutical cationsalts.

The non-self-immolative linker component is one of the followingstructures:

Wherein the (*) atom is the point of attachment of additional spacer orreleasable linkers, the cytotoxic agents, and/or the binding molecules;X¹, Y¹, Q¹, R₁₂, R₁₂′ are defined as above; r is 0˜100; p and q are 0˜6independently.

Spacer (T) is a linear, branched or cyclic alkyl, alkenyl, alkynyl oraryl having from 1 to 10 carbon atoms, or T can be a polyethylene glycol(—CH₂CH₂O—) spacer; t is 0, or 1˜100. T can also undergo cyclizationupon amide bond hydrolysis, such amides include substituted andunsubstituted 4-aminobutyric acid amides, appropriately substitutedbicycle [2.2.1] and bicyclo[2.2.2] ring systems, and2-aminophenylpropionic acid amides.

Q is a cell-binding agent/molecule (CBA), or a functional group thatenables linkage with a cell-binding agent, or a functional group thatenables linkage with a linker attached on a cell-binding agent. Thefunction group is chosen from a thiol, an amine, a hydrazine, analkoxylamino, a disulfide substituent, a maleimido, a haloacetyl group,a carboxy acid, an N-hydroxy succinimide ester, a ketone, an ester, analdehyde, an alkynyl, an alkenyl, or a protected thiol or disulfidegroup, such as SAc, SSR₁ or SSAr. Ar is an aromatic group or heteroaromatic group.

In a further aspect, the present invention discloses a therapeuticcomposition comprising: (1). an effective amount of one or morederivatives of amanita toxins optionally linkable or linked to a cellbinding agent, and (2). a pharmaceutically acceptable carrier, diluent,or excipient, of Formula (I) of the patent application, to kill targetcells or tissues containing target cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of dipeptide Tr-Hpi-Gly-OH.

FIG. 2 shows the solution phase synthesis of Ile-S-deoxo-amanitin_1.

FIG. 3 shows the solution phase synthesis of Ile-S-deoxo-amanitin_2.

FIG. 4 shows the solid phase synthesis of Ile³-S-deoxo-amanitinprecursor.

FIG. 5 shows the synthesis of Ile³-S-deoxo-amanitin derivatives_1.

FIG. 6 shows the synthesis of linker_1 for the synthesis of amanitatoxins.

FIG. 7 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_2.

FIG. 8 shows the synthesis of linker_2 for the synthesis of amanitatoxins.

FIG. 9 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_3.

FIG. 10 shows the synthesis of linker_3 for the synthesis of amanitatoxins.

FIG. 11 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_4.

FIG. 12 shows the synthesis of Synthesis of linker_4 of amanita toxins.

FIG. 13 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_5

FIG. 14 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_6.

FIG. 15 shows the synthesis of linker_5 of amanita toxins.

FIG. 16 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_7.

FIG. 17 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_8.

FIG. 18 shows the synthesis of linker_6 for amanita toxins.

FIG. 19 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_9.

FIG. 20 shows the synthesis of Ile³-S-deoxo-amanitin derivatives andconjugates_10.

FIG. 21 shows the synthesis of conjugates of amanitin derivatives_1.

FIG. 22 shows the synthesis of conjugates of amanitin derivatives_2.

FIG. 23 shows the synthesis of conjugates of amanitin derivatives_3.

FIG. 24 shows the synthesis of conjugates of amanitin derivatives_4.

FIG. 25 shows the synthesis of conjugates of phalloidin derivatives_1.

FIG. 26 shows the synthesis Solution phase synthesis of amatoxin andderivatives.

FIG. 27 shows the solution phase synthesis of phallotoxin andderivatives.

FIG. 28 shows the solution phase synthesis of virotoxin and derivatives.

FIG. 29 shows the comparison of the anti-tumor effect of conjugatecompounds A1, A2, A3, A5, A6, A7, A9, and A10 with T-DM1 using humangastric tumor N87 cell model at dosing of 6 mg/kg, i.v., one injection.It shows all the conjugates did not cause the animal body weight loss

FIG. 30 shows the comparison of the anti-tumor effect of conjugatecompounds A1, A2, A3, A5, A6, A7, A9, and A10 with T-DM1 using humangastric tumor N87 cell model at dosing of 6 mg/kg, i.v., one injection.The animals at control group were sacrificed at day 45. All the 8conjugate compounds (A1, A2, A3, A5, A6, A7, A9, and A10) had better invivo activity than T-DM1.

FIGS. 31A to 31F are exemplary ADCs.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched having 1 to 8 carbon atoms in the chain or cyclic. “Branched”means that one or much lower alkyl groups such as methyl, ethyl orpropyl are attached to a linear alkyl chain. Exemplary alkyl groupsinclude methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl,3-pentyl, octyl, nonyl, decyl, cyclopentyl, cyclohexyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁˜C₈ alkyl, —O—(C₁˜C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen (F, Cl, Br or I), —N₃, —NH₂, —NH(R′), —N(R′) 2 and —CN;where each R′ is independently selected from —C₁˜C₈ alkyl and aryl.

A “cyclic alkyl”, “cycloalkyl” and “C₃˜C₈ carbocycle” can be usedinterchangeably. They mean a 3-, 4-, 5-, 6-, 7- or 8-membered saturatedor unsaturated non-aromatic carbocyclic ring. Representative C₃˜C₈carbocycles include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl,1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, andcyclooctadienyl. A C₃˜C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁˜C₈ alkyl, —OR₅, -aryl, —C(O)R₅, —OC(O)R₅, —C(O)OR₅, —C(O)NH₂,—C(O)NHR₅, —C(O)NR₅R_(5′)—NHC(O)R₅, —S(O)₂R₅, —S(O)R₅, —OH, -halogen,—N₃, —NH₂, —NHR₅, —NR₅R_(5′) and —CN; wherein R₅ and R_(5′) areindependently H; C₁˜C₈ of alkyl, alkenyl, alkynyl, heteroalkyl, aryl,arylalkyl, or carbonylalkyl; or pharmaceutical salts. R₅ and R_(5′) canfurther be substituted with at least one substituent selected from˜N(R₅)(R₅), —CO₂H, —SO₃H, —OR₅, —CO₂R₅, —CONKS, and —PO₃H.

A “C₃˜C₈ carbocyclo” refers to a C₃˜C₈ carbocycle group defined abovewherein one of hydrogen atoms on the carbocycle is replaced with a bond.

Alkenyl refers to an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched having 2to 8 carbon atoms in the chain. The alkenyl double bond may have “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.Exemplary alkenyl groups include, but are not limited to, ethylenyl orvinyl, propenyl or allyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl,n-pentenyl, hexylenyl, heptenyl, octenyl.

“Alkynyl” or “alkinyl” means an aliphatic hydrocarbon group containing acarbon-carbon triple bond and which may be straight or branched having 2to 8 carbon atoms in the chain. Exemplary alkynyl groups includeethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, pentynyl,n-pentynyl, hexylynyl, heptynyl, and octynyl.

“Heteroalkyl” is C₂˜C₈ alkyl in which one to four carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N.

“Heterocycle” refers to an aromatic or non-aromatic C₃˜C₁₄ carbocycle inwhich one to four of the ring carbon atoms are independently replacedwith a heteroatom from the group of O, N, P, S and Se. Preferableheteroatoms are oxygen, nitrogen and sulphur. Suitable heterocyclics arealso disclosed in The Handbook of Chemistry and Physics, 76^(th)Edition, CRC Press, Inc., 1995-1996, p 2-25 to 2-26, the disclosure ofwhich is hereby incorporated by reference. Preferred non aromaticheterocyclic include, but are not limited to pyrrolidinyl,pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl,tetrahydro-pyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl,morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl,thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl,dihydro-pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydro-pyridyl,dihydropyridyl, tetrahydropyrmidinyl, dihydrothiopyranyl, azepanyl, aswell as the fused systems resulting from the condensation with a phenylgroup.

“Aryl” or Ar refers to an aromatic or hetero aromatic group, composed ofone or several rings, comprising three to fourteen carbon atoms,preferentially six to ten carbon atoms. The term of hetero aromaticgroup refers one or several carbon on aromatic group, preferentiallyone, two, three or four carbon atoms are replaced by O, N, Si, Se, P orS, preferentially by O, S, N. The term aryl or Ar also refers to aaromatic group, wherein one or several H atoms are replacedindependently by alkyl, F, Cl, Br, I, OR₅, or SR₅, NR₅R_(5′), N═NR₅,N═R₅, NR₅R_(5′), NO₂, SOR₅R₅, SO₂R₅, SO₃R₅, OSO₃R₅, PR₅R_(5′),POR₅R_(5′), PO₅R_(5′), OPO₃R₅R_(5′), or PO₃R₅R_(5′) wherein R₅ andR_(5′) are independently H, alkyl, alkenyl, alkinyl, heteroalkyl, aryl,arylalkyl, carbonyl, or pharmaceutical salts.

The term “heteroaryl” or aromatic heterocycles refers to a 5 to 14,preferably 5 to 10 membered aromatic hetero, mono-, bi- or multicyclicring. Examples include pyrrolyl, pyridyl, pyrazolyl, thienyl,pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl,imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl,1,2,4-thiadiazolyl, isothiazolyl, triazoyl, tetrazolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl,isoxazolyl, pyridyl-N-oxide, as well as the fused systems resulting fromthe condensation with a phenyl group.

“Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl”, “heteroaryl”,“heterocyclic” and the like refer also to the corresponding “alkylene”,“cycloalkylene”, “alkenylene”, “alkynylene”, “arylene”, “heteroarylene”,“heterocyclene” and the likes which are formed by the removal of twohydrogen atoms.

“Halogen atom” refers to fluorine, chlorine, bromine or iodine atom;preferably bromine and chlorine atom.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

“Pharmaceutically acceptable excipient” includes any carriers, diluents,adjuvants, or vehicles, such as preserving or antioxidant agents,fillers, disintegrating agents, wetting agents, emulsifying agents,suspending agents, solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions as suitabletherapeutic combinations.

As used herein, “pharmaceutical salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, tartaric, citric, methanesulfonic, benzenesulfonic,glucoronic, glutamic, benzoic, salicylic, toluenesulfonic, oxalic,fumaric, maleic, lactic and the like. Further addition salts includeammonium salts such as tromethamine, triethanolamine, meglumine,epolamine, etc., metal salts such as sodium, potassium, calcium, zinc ormagnesium.

The pharmaceutical salts of the present invention can be synthesizedfrom the parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreaction of the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. Generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p 1418, the disclosure of which is hereby incorporated byreference.

The term “compound”, “cytotoxic agent”, “cytotoxic compound,” “cytotoxicdimer” and “cytotoxic dimer compound” are used interchangeably. They areintended to include compounds for which a structure or formula or anyderivative of thereof has been disclosed in the present invention or astructure or formula or any derivative thereof that has beenincorporated by reference. The term also includes, stereoisomers,geometric isomers, tautomers, solvates, metabolites, salts (e.g.,pharmaceutically acceptable salts) and prodrugs, and prodrug salts of acompound of all the formulae disclosed in the present invention. Theterm also includes any solvates, hydrates, and polymorphs of any of theforegoing. The specific recitation of “stereoisomers,” “geometricisomers,” “tautomers,” “solvates,” “metabolites,” “salt” “prodrug,”“prodrug salt,” “conjugates,” “conjugates salt,” “solvate,” “hydrate,”or “polymorph” in certain aspects of the invention described in thisapplication shall not be interpreted as an intended omission of theseforms in other aspects of the invention where the term “compound” isused without recitation of these other forms.

“Cell binding agents” or “Cell binding molecules” may be of any kindpresently known, or those become known, and include peptides andnon-peptides. Generally, these can be antibodies (especially monoclonalantibodies) or a fragment of an antibody that contains at least onebinding site, lymphokines, hormones, growth factors, nutrient-transportmolecules (such as transferrin), or any other cell binding molecule orsubstance (such as vitamins).

More specific examples of cell binding agents that can be used include:

-   -   monoclonal antibodies (mAb);    -   single chain antibodies;    -   fragments of antibodies such as Fab, Fab′, F(ab′)₂, F_(v),        (Parham, J. Immunol. 131, 2895-2902 (1983); Spring et al, J.        Immunol. 113, 470-478 (1974); Nisonoff et al, Arch. Biochem.        Biophys. 89, 230-244 (1960)), fragments produced by a Fab        expression library, anti-idiotypic (anti-Id) antibodies, CDR's,        and epitope-binding fragments of any of the above which        immunospecifically bind to cancer cell antigens, viral antigens        or microbial antigens.    -   interferons;    -   peptides; or conjugated proteins or peptides;    -   lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6;    -   hormones such as insulin, TRH (thyrotropin releasing hormones),        MSH (melanocyte-stimulating hormone), steroid hormones, such as        androgens and estrogens;    -   growth factors and colony-stimulating factors such as EGF, TGFα,        insulin like growth factor (IGF-I, IGF-II) G-CSF, M-CSF and        GM-CSF (Burgess, Immunology Today 5, 155-158 (1984)); vitamins,        such as folate and    -   transferrin (O'Keefe et al, J. Biol. Chem. 260, 932-937 (1985)).

Monoclonal antibodies (mAb), mAb single chain or fragments can beproduced in the well known state of art technology. The technologypermits the production of extremely selective cell binding agents in theform of specific monoclonal antibodies. The well known in the art aretechniques for creating monoclonal antibodies produced by immunizingmice, rats, hamsters or any other mammal with the antigen of interestsuch as the intact target cell, antigens isolated from the target cell,whole virus, attenuated whole virus, and viral proteins such as viralcoat proteins.

Selection of appropriate cell binding agents is a matter of choice thatdepends upon the particular cell population that is to be targeted, butin general monoclonal antibodies are preferred if an appropriate one isavailable.

For example, an anti-CD20 antigen monoclonal antibody, known asRituximab is a chimeric (mouse/human) monoclonal antibody and it was thefirst therapeutic antibody approved by the United States Food and DrugAdministration for treatment of relapsed or refractory low-grade orfollicular NHL (Leonard, J. P. et al., Clin. Canc. Res. 10:5327-5334(2004)). Another anti-CD20 antibody, known as Ofatumumab, is a humanmonoclonal antibody targeting an epitope different from that ofrituximab and most other CD20 directed antibodies. It was approved by USFDA for treating chronic lymphocytic leukemia and has also shownpotential in treating Follicular non-Hodgkin's lymphoma, Diffuse large Bcell lymphoma, rheumatoid arthritis and relapsing remitting multiplesclerosis (Coiffier, B. et al Blood 111: 1094-100 (2008); Zhang, B. MAbs1 (4): 326-31 (2009)). A third-generation, humanized andglyco-engineered anti-CD20 mAb for the treatment of B-cell lymphoidmalignancies named Afutuzumab (now called obinutuzumab) has beendeveloped (Robak, T (2009) Current opinion in investigational drugs(London, England: 2000) 10 (6): 588-96). Obinutuzumab is fully humanizedIgG1 type II anti-CD20 antibody and it selectivity binds to theextracellular domain of the human CD20 antigen on malignant human Bcells. Similarly, an anti-CD19 antigen monoclonal antibody B4 is amurine IgG₁, that binds to the CD19 antigen on B cells (Nadler et al,131 J. Immunol. 244-250 (1983)) and can be used if the target cells areB cells or diseased cells that express CD19 antigen such as innon-Hodgkin's lymphoma or chronic lymphoblastic leukemia. In addition,the anti-CD22 antibodies that include RFB4 (Mansfield, E. et al., Blood90:2020-2026 (1997)), CMC-544 (DiJoseph, J. F., Blood 103:1807-1814(2004)) and LL2 (Pawlak-Byczkowska, E. J. et al., Cancer Res.49:4568-4577 (1989)) can be used as potential therapies for B cellcancers and other B cell proliferative diseases. The LL2 antibody(formerly called HPB-2) is an IgG2a mouse monoclonal antibody directedagainst the CD22 antigen (Pawlak-Byczkowska, E. J. et al. (1989),supra). Furthermore, the anti CD33 antigen monoclonal antibody, namedGemtuzumab was first monoclonal antibody conjugated with a cytotoxicdrug to treat acute myelogenous leukemia (AML) (P. F. Bross et al ClinCancer Res 7 (6): 1490-6). A similar anti CD33 antigen antibody, namedMy9-6 is a murine IgG₁ antibody that binds specifically to the CD33Antigen (J. D. Griffin et al 8 Leukemia Res., 521 (1984)) and can beused to target cells express CD33 as in the disease of acute myelogenousleukemia (AML). Additionally, GM-CSF antibody which binds to myeloidcells can be used as a cell binding agent to diseased cells from acutemyelogenous leukemia. IL-2 antibody, which binds to activated T-cells,can be used for prevention of transplant graft rejection, for therapyand prevention of graft-versus-host disease, and for the treatment ofacute T-cell leukemia. MSH antibody, which binds to melanocytes, can beused for the treatment of melanoma.

Novel Cytotoxic Agents and their Conjugation of the Invention.

The novel amanita toxin derivatives according to the present inventioncomprises one or more derivatives of amatoxins, phallotoxins orvirotoxins, optionally linkable or linked to a cell binding agent via alinking group. The linking group is part of a chemical moiety that iscovalently bound to amanita toxin derivatives through conventionalmethods.

The amanita toxin derivatives disclosed in the present invention havethe formula (I) shown below:

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers.

Wherein

---- represents a single bond that links any carbon position of thearomatic (indole) ring;

represents an optional single bond or an absent bond.

R₁ and R₂ are independently selected from H, OH, CH₂OH, CH(OH)CH₂OH,CH(CH₃)CH₂OH, CH(OH)CH₃, C₁-C₈ alkyl, —OR₁₂ (ether), C₂-C₈ alkenyl,alkynyl, heteroalkyl, —OCOR₁₂ (ester), —OC(═O)OR₁₂-(carbonate),—OC(═O)NHR₁₂(carbamate); C₃-C₈ aryl, heterocyclic, carbocyclic,cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl.

R₃ and R₄ are independently selected from H, OH, —OR₁₂ (ether), —OCOR₁₂(ester), —OCOCH₃ (acetate), —OCOOR₁₂ (carbonate),—OC(═O)NHR₁₂(carbamate), —OP(O)(OR₁₂)(OR₁₂′) (phosphate),OP(O)(NHR₁₂)(NHR₁₂′) (phosphamide), O—SO₃ ⁻, or O-glycoside.

R₅ is selected from H, OH, NH₂, —OR₁₂, —NHR₁₂, —NR₁₂R₁₂′,N(H)(R₁₂)R₁₃CO(Aa)_(p), (an amino acid or peptide, wherein Aa is anamino acid or a polypeptide, p represents 0-6).

R₆ is selected from H, OH, CH₂OH, CH(OH)CH₂OH, CH(CH₂OH)₂, CH(CH₃)OH,CH₂CH₂OH, PrOH, BuOH, C₁˜C₈ alkyl, —OR₁₂ (ether), C₂˜C₈ alkenyl,alkynyl, heteroalkyl, —OCOR₁₂ (ester); C₃˜C₈ aryl, heterocyclic, orcarbocyclic.

R₇, R₈ and R₉ are independently selected from H, OH, CH₃, CH(CH₃)₂,CH(CH₃)CH₂CH₃, CH₂OH, CH(OH)CH₂OH, CH₂CH(OH)CH₂OH, CH(CH₂OH)₂,CH₂C(OH)(CH₂OH)₂, CH₂C(OH)(CH₃)(CH₂OH), CH₂C(OH)(CH(CH₃)₂)(CH₂OH),CH₂CH₂OH, PrOH, BuOH, CH₂COOH, CH₂CH₂COOH, CH(OH)COOH, CH₂CONH₂,CH₂CH₂CONH₂, CH₂CH₂CH₂CH₂NH₂, CH₂CH₂CH₂NHC(═NH)NH₂, C₁˜C₈ alkyl, CH₂Ar,CH₂SH, CH₂SR₁₂, CH₂SSR₁₂, CH₂SSAr, CH₂CH₂SCH₃, —OR₁₂ (ether), C₂˜C₈alkenyl, alkynyl, heteroalkyl, —OCOR₁₂ (ester); C₃˜C₈ aryl,heterocyclic, or carbocyclic.

R₁₀ is selected from H, NH₂, OH, SH, NO₂, halogen, —NHOH, —N₃ (azido);—CN (cyano); C₁˜C₈ alkyl, C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈aryl, heterocyclic, or carbocyclic; —OR₁₂ (ether), —OCOR₁₂ (ester),—OCOCH₃ (acetate), —OC(O)OR₁₂ (carbonate), —OC(O)CH(R₁₂)NHAa (Aa is anamino acid group), —NR₁₂R₁₂′(amine), —NR₁₂COR₁₂′(amine), —NR₁₂NR₁₂′NR₁₂″(amine); —OCONR₁₂R₁₂′(carbamate); —NR₁₂(C═NH)NR₁₂′R₁₂″ (guanidinum);—NR₁₂CO(Aa)_(p), (an amino acid or peptide, wherein Aa is an amino acidor a polypeptide, p represents 0-6); —N(R₁₂)CONR₁₂′R₁₂″ (urea);—OCSNHR₁₂ (thiocarbamate); —SH (thiol); —SR₁₂ (sulfide); —S(O)R₁₂(sulfoxide); —S(O₂)R₁₂ (sulfone); —SO₃, HSO₃, HSO₂, or a salt of HSO₃ ⁻,SO₃ ²⁻ or —HSO₂ ⁻ (sulphite); —OSO₃ ⁻; —N(R₁₂)SOOR₁₂′ (sulfonamide);H₂S₂O₅ or a salt of S₂O₅ ²⁻ (metabisulfite); PO₃SH₃, PO₂S₂H₂, POS₃H₂,PS₄H₂ or a salt of PO₃S³⁻, PO₂S₂ ³⁻, POS₃ ³⁻, PS₄ ³⁻ (mono-, di-, tri-,and tetra-thiophosphate); (R₁₂O)₂POSR₁₂′ (thiophosphate ester); HS₂O₃ ora salt of S₂O₃ ²⁻ (thiosulfate); HS₂O₄ or a salt of S₂O₄ ²⁻(dithionite); (P(═S)(OR₁₂)(S)(OH) or a salt formed with a cation(phosphorodithioate); —N(R₁₂)OR₁₂′ (hydroxylamine derivative);R₁₂C(═O)NOH or a salt formed with a cation (hydroxamic acid);(HOCH₂SO₂′, or its salts (formaldehyde sulfoxylate); —N(R₁₂)COR₁₂′(amide); R₁₂R₁₂′R₁₂″NPO₃H (trialkylphosphoramidate or phosphoramidicacid); or ArAr′Ar″NPO₃H (triarylphosphonium); OP(O)(OM₁)(OM₂),OCH₂OP(O)(OM₁)(OM₂), OSO₃M₁; O-glycoside (glucoside, galactoside,mannoside, glucuronoside, alloside, fructoside, etc), NH-glycoside,S-glycoside or CH₂-glycoside; M₁ and M₂ are independently H, Na, K, Ca,Mg, NH₄, NR₁′R₂′R₃′; R₁′, R₂′ and R_(3′) are independently H, C₁˜C₈alkyl; Ar, Ar′, and Ar″ are C₃˜C₈ aryl or heteroaromatic group.

R₁₁ is H, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl,heteraoaryl.

R₁₂, R₁₂′, and R₁₂″ are independently selected from H, C₁˜C₈ alkyl;C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl, heteraoaryl,heterocyclic, or carbocyclic.

X is S, O, NH, SO, SO₂, or CH₂.

m is 0 or 1, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20.

L is a linker or a linker-cell-binding molecule (Q) covalently boundcluster, or a linker which has a functional group on the linker thatenables linkage with a cell-binding agent (CBA). L is preferably areleasable linker, which has the formula of: -Ww-(Aa)r-Tt-; or-Ww-(Aa)r-Tt-Q; or Q-Ww-(Aa)r-Tt-; wherein W is a Stretcher unit; w is 0or 1; Aa is an Amino Acid unit comprising independent amino acids; r isan integer ranging from 0 to 100. The Stretcher unit W may contain aself-immolative or a non-self-immolative component, peptidyl units, ahydrazone bond, a disulfide, an ester, an oxime, an amide, or athioether bond. The self-immolative unit includes, but is not limitedto, aromatic compounds that are electronically similar to thepara-aminobenzylcarbamoyl (PAB) groups such as2-aminoimidazol-5-methanol derivatives, heterocyclic PAB analogs,beta-glucuronide, and ortho or para-aminobenzylacetals. Preferably, theself-immolative linker component has one of the following structures:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker units, or the cytotoxic agent, and/or the bindingmolecule (CBA); X¹, Y¹, Z² and Z³ are independently NH, O, or S; Z¹ isindependently H, NH, O or S; v is 0 or 1; Q′ is independently H, OH,C₁˜C₆ alkyl, (OCH₂CH₂)_(n), F, Cl, Br, I, OR₁₂, SR₁₂, NR₁₂R₁₂′, N═NR₁₂,N═R₁₂, NR₁₂R₁₂′, NO₂, SOR₁₂R₁₂′, SO₂R₁₂, SO₃R₁₂, OSO₃R₁₂, PR₁₂R₁₂′,PO₂R₁₂R₁₂′, OPO(OR₁₂)(OR₁₂′), or OCH₂PO(OR₁₂(OR₁₂′) wherein R₁₂ and R₁₂′are as defined above; preferably R₁₂ and R₁₂′ are independently selectedfrom H, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl,heterocyclic, carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl,alkylcarbonyl; or pharmaceutical cation salts.

The non-self-immolative linker component is one of the followingstructures:

Wherein the (*) atom is the point of attachment of additional spacer orreleasable linkers, the cytotoxic agents, and/or the binding molecules;X¹, Y¹, Q¹, R₁₂, R₁₂′ are defined as above; r is 0˜100; p and q are 0˜6independently.

Spacer (T) is a linear, branched or cyclic alkyl, alkenyl, alkynyl oraryl having from 1 to 10 carbon atoms, or T can be a polyethylene glycol(—CH₂CH₂O—) spacer; t is 0, or 1˜100. T can also undergo cyclizationupon amide bond hydrolysis, such amides include substituted andunsubstituted 4-aminobutyric acid amides, appropriately substitutedbicycle [2.2.1] and bicycle [2.2.2] ring systems, and2-aminophenylpropionic acid amides.

The linker L can be also selected from the group consisting of: R₁₂,OR₁₂, OR₁₂O, NHR₁₂, NHR₁₂NH, NR₁₁R₁₂, SR₁₂S, OR₁₂NH, OR₁₂Ar, NHR₁₂Ar,—(CR₁₁R₁₄-(Aa)_(r)(CR₁₂′R₁₂′)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)(Aa)_(r)(OCH₂CH₂)_(t)—,-(Aa)_(r)-(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(n)(OCH₂CH₂)_(t)(Aa)_(r)-,—(CR₁₁R₁₂)_(p)(CH═CH)(CR₁₂′R₁₂″)_(q)(Aa)_(r)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(NR_(12′)CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(Aa)_(t)(NHCO)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(r)—,(CR₁₁R₁₂)_(p)(OCO)(Aa)_(r)-(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)—(CO)-(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(NR₁₁CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)—(OCO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)-phenyl-CO-(Aa)_(r)(CR₁₂′R₁₂′)_(q),—(CR₁₁R₁₂)_(p)-furyl-CO-(Aa)_(t)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-oxazolyl-CO-(Aa)_(r)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-thiazolyl-CO-(Aa)_(t)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-thienyl-CO—(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-imidazolyl-CO—(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)-morpholino-CO-(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)-piperazino-CO(Aa)_(r)-(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)—N-methyl-piperazin-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)(Aa)_(r)-phenyl-, —(CR₁₁R₁₂)_(p)-(Aa)_(r)-furyl-,—(CR₁₁R₁₂)_(p)-oxazolyl(Aa)_(r)-, —(CR₁₁R₁₂)_(p)-thiazolyl-(Aa)_(r)-,—(CR₁₁R₁₂)_(p)-thienyl-(Aa)_(t)-, —(CR₁₁R₁₂)_(p)-imidazolyl(Aa)_(r)-,—(CR₁₁R₁₂)_(p)-morpholino-(Aa)_(r)-,—(CR₁₁R₁₂)_(p)-piperazino-(Aa)_(t)-,—(CR₁₁R₁₂)_(p)—N-methylpiperazino-(Aa)_(t)-,—K(CR₁₁R₁₂)_(p)-(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)(Aa)_(r)(OCH₂CH₂)_(t)—,—K(Aa)_(r)(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(r)(Aa)_(t),—K(CR₁₁R₁₂)_(p)(CR₇═CR₈)(CR₁₂′R₁₂″)_(q)-(Aa)_(r)(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(NR₇CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)-(Aa)_(t)(NR₇—CO)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(OCO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—K(—CR₁₁R₁₂)_(p)(NR₁₁CO)(Aa)_(r)(CR₁₁′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(OCO)-(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂N,—K(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),R₁₂)_(p)(CO)(Aa)_(r)-, —K(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(r)Q,—K(CR₁₁R₁₂)_(p)-phenyl-CO-(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-furyl-CO(Aa)_(r)-(CR₁₂′R₁₂″)_(q),—K(C₁₁R₁₂)_(p)-oxazolyl-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-thiazolyl-CO(Aa)_(t)-(CR₁₂′R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)-thienyl-CO(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-imidazolyl-CO—(CR₁₂′R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)-morpholino-CO(Aa)_(t)(CR₁₂′R₁₂″)_(q)—,—K(C₁₁R₁₂)_(p)-piperazino-CO-(Aa)_(r)(CR₁₂′R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)—N-methylpiperazin-CO(Aa)_(r)-(CR₁₂′R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)-(Aa)_(r)-phenyl-, —K(CR₁₁R₁₂)_(m)(Aa)_(r)-furyl-,—K(CR₁₁R₁₂)_(p)-oxazolyl(Aa)_(r)-, —K(CR₁₁R₁₂)_(m)-thiazolyl-(Aa)_(r)-,—K(CR₁₁R₁₂)_(p)-thienyl-(Aa)_(r), —K(CR₁₁R₁₂)_(p)-imidazolyl(Aa)_(r)-,—K((CR₁₁R₁₂)_(m)-morpholino-(Aa)_(r),—K(CR₁₁R₁₂)_(p)-piperazino-(Aa)_(t)-,—K(CR₁₁R₁₂)_(m)N-methylpiperazino-(Aa)_(r).

Wherein Aa, r, n, p, q, t, R₇, R₁₁, R₁₂, R₁₂′, R₁₂″ are as definedabove. K is NR₁₂, O, S, Se, B, C₃˜C₁₀ of Ar or Heterocyclic.

Q is a cell-binding molecule (CBA), or a functional group that enableslinkage with a cell-binding agent, or a functional group that enableslinkage with a linker attached on a cell-binding agent. The functiongroup is chosen from a thiol, an amine, a hydrazine, an alkoxylamino, adisulfide substituent, a maleimido, a haloacetyl group, a carboxy acid,an N-hydroxy succinimide ester, a ketone, an ester, an aldehyde, analkynyl, an alkenyl, or a protected thiol or disulfide group, such asSAc, SSR₁ or SSAr. Ar is an aromatic group or hetero aromatic group.

The compounds of the general formula (I) having geometrical andstereoisomers are also a part of the invention.

In certain embodiments, the amanita toxin derivatives are presented bythe following formula (Ia) (Ib), and (Ic), wherein an amide linkerlinked to C-5 position of the indole unit.

Wherein “----”, “

”, R₁, R₂, R₄, R₅, R₇, R₈, R₉, R₁₀, L and Q are defined the same as inFormula (I).

In certain embodiments, the amanita toxin derivatives are presented bythe following formula (Id), wherein an amide linker linked to C-7position of the indole unit.

Wherein R₁, R₂, R₄, R₅, R₁₀, L and Q are defined the same as in Formula(I).

In certain embodiments, the amanita toxin derivatives of formula (Ia),(Ib), and (Ic) are represented by the following formulas (Ia-1), (Ia-2),(Ia-3), (Ia-4), (Ia-5), (Ia-6), (Ia-7), (Ia-8), (1a-9), (Ia-10),(Ia-11), (Ia-12), (Ia-13), (Ia-14), (Ia-15), (Ia-16), (Ia-17), (Ia-18),(Ia-19), (Ia-20), (Ia-21), (Ia-22), (Ia-23), (Ia-24), (Ib-1), (Ib-2),(Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ib-7), (Ic-1), (Ic-2), (Ic-3), (Ic-4),(Ic-5), and (Ic-6):

Wherein R₁₀, L and Q are defined the same as in Formula (I).

In certain embodiments, the amanita toxin derivatives of formula (Id)are represented by the following formulas (Id-1), (Id-2), (Id-3),(Id-4), (Id-5), (Id-6), (Id-7), (Id-8), (Id-9), (Id-10), (Id-11),(Id-12), (Id-13), (Id-14), (Id-15), (Id-16), (Id-17), (Id-18), (Id-19),(Id-20), and (Id-21):

Wherein R₁₄ is H, PO₃ ²″, SO₃ ⁻, R₁₂, —COR₁₂, —COCH₃, —COOR₁₂,—CONR₁₂R₁₂′, —C(═O)R₁₂NH(Aa)_(t), (an amino acid or peptide, wherein Aais an amino acid or a polypeptide, t represents 0-100); —CSNHR₁₂(thiocarbamate); —SOR₁₂ (sulfoxide); —SO₂R₁₂ (sulfone); —SO₃ ⁻, HSO₃,HSO₂, or a salt of HSO₃ ⁻, SO₃ ²⁻ or —HSO₂ ⁻(sulphite); P(O)(OM₁)(OM₂),CH₂OP(O)(OM₁)(OM₂), SO₃M₁; glycoside (glucoside, galactoside, mannoside,glucuronoside, alloside, fructoside, etc), M₁ and M₂ are independentlyH, Na, K, Ca, Mg, NH₄, NR₁′R₂′R₃′; R₁′, R₂′ and R_(3′) are independentlyH, C₁˜C₈ alkyl.

In further embodiments, the cytotoxic agent and its conjugate of thisinvention are preferred one of the following structures:

or a pharmaceutically acceptable salt, hydrates, or hydrated salt, orthe polymorphic crystalline structure of the compounds thereof, or theoptical isomers, racemates, diastereomers or enantiomers.

Wherein Aa, r, n, L and Q are the same in the Formula (I). PEG ispolyethylene glycol with the formula of —OCH₂CH₂. Preferably, Q is H,C₁˜C₈ of alkyl, alkenyl, alkinyl, aryl, cyclic, cyclohetero, haloalkyl,alkoxy, haloalkoxy alkylamino; or halogen; or —NO₂; or —CN; —SH; —SSCH₃;—SSAc; —SSAr; —SS-Pyridine; —SS-Ar(NO₂); —S-cell binding agent; or afunction group of NHS ester, pentafluorophenyl ester; alkyloxyamine;aldehyde; ketone; carboxyl acid; hydrazine; amine; or thiolactone; orlinked a cell binding agent via Stretcher units (Ww) or via Spacer units(Tt), wherein W, w, T, and t are as defined in Formula (I); or Q isselected from any one of the following formulas:

Wherein D is H, —NO₂, SO₃ ⁻, CN, or F; R₁, R₂, R₃, R₄, r, m, and n aredescribed in Formula (I); w and w′ are 0 or 1 independently.

Synthesis of the Derivatives of Amanita Toxins as Cytotoxic Agents.

The compounds and process of the present invention can be prepared in anumber of ways well known to those skilled in the art. The compounds canbe synthesized, for example, by application or adaptation of the methodsdescribed in the examples, or variations thereon as appreciated by theskilled artisan. The appropriate modifications and substitutions will bereadily apparent and well known or readily obtainable from thescientific literature to those skilled in the art. In particular, suchmethods can be found in Richard C. Larock, “Comprehensive OrganicTransformations, A Guide to Functional Group Preparations”, Two VolumeSet, 2nd Edition, Wiley Publishers, 2010.

One preferred aspect of preparation of theses derivatives of Amanitatoxins of the invention is semi-synthesis. Thus, the core structurecompounds of theses derivatives of Amanita toxins are isolated fromtheses derivatives-containing mushroom species from the genera Amanita,Galerina and Lepiota, in particular, A. bisporigera, A. visosa, A.suballiacea, A. phalloides and allied species (Hallen, H. E. et al, 2007Proc. Nat. Acad. Sci. USA, 104, 19097-101), or isolated fromfermentation using a basidiomycete, or using A. fissa (Guo, X. W., etal, 2006 Wei Sheng Wu Xue Bao 46(3): 373-8). The isolated toxincompounds are sequentially undergone aromatic nitration of the indoleunits, followed by reduction of the nitro group to an amine, and thenthe produced amine compounds are condensated with a linker having areactive or a reactable carboxylic group to form an amide linkage. Anillustration of the semi-synthesis steps are shown below:

Wherein R₁₀, L and Q are defined the same in the Formula (I). Wherein Lvis leaving group selected from OH, halogen, NHS (N-hydroxylsuccinimide), nitrophenol, pentalfluorophenol, etc.

The nitrition above can be using nitric acid, or mixture of nitric acidand sulfuric acids, or mixture of nitric acid and acetic acid optionallycontaining acetic anhydride, or tert-Butyl nitrite (TBN), or nitrosoniumsalts (NO⁺BF₄ ⁻, NO⁺ClO₄, NO⁺PF⁻ ₆, NO⁺AsF⁻ ₆, NO⁺SbF⁻ ₆), or nitroniumsalts (NO²⁺BF⁴⁻, NO²⁺ClO⁴⁻, NO²⁺BF⁻⁶, NO²⁺AsF⁻⁶, NO²⁺SbF⁻⁶) Thereduction of the nitro group to the amine can be by many different ways,such as catalytic hydrogenation reduction using palladium-on-carbon,platinum(IV) oxide, or Raney nickel; or direct reduction by using ironin acidic media, sodium hydrosulfite; sodium sulfide (or hydrogensulfide and base), tin(II) chloride, titanium(III) chloride,triphenylphosphine, trialkylphosphine, zinc, or samarium. The finalcondensation of the amino group to the linker L containing a carboxylicacid derivative, —C(O)-Lv. Wherein Lv can be selected from F, Cl, Br, I,nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol;pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol;pentachlorophenol; triflate; imidazole; dichlorophenol;tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate;2-ethyl-5-phenylisoxazolium-3′-sulfonate, anhydrides formed its self, orformed with the other anhydride, e.g. acetyl anhydride, formylanhydride. When Lv is OH group, then the condensation with the aminogroup is through a coupling reagent, such as a peptide coupling agent,or coupling agent for Mitsunobu reaction. These coupling reagents are,but not limited: N-(3-dimethylamino-propyl)-N′-ethylcarbodiimide (EDC),dicyclohexyl-carbodiimide(DCC), N,N′-diisopropyl-carbodiimide (DIC),N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate(CMC, or CME-CDI), 1,1′-carbonyldiimidazole (CDI),O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU),benzotriazol-1-yloxy)tris(dimethyl-amino)phosphonium hexafluorophosphate(BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC),chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate,1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino)methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxidehexafluorophosphate (HDMA), 2-chloro-1,3-di methyl imidazolidiniumhexafluorophosphate (CIP), chlorotripyrrolidinophosphoniumhexafluorophosphate (PyCloP),fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate(BTFFH), N,N,N′,N′-tetramethyl-S-(1-oxido-2-pyridyl)thiuroniumhexafluorophosphate, O-(2-oxo-1(2H)pyridyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TPTU),S-(1-oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate,O-[(ethoxycarbonyl) cyano-methylenamino]-N,N,N,N′-tetramethyluroniumhexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate(COMU),O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (HBPyU), N-benzyl-N′-cyclohexylcarbodiimide (with,or without polymer-bound), dipyrrolidino(N-succinimidyloxy)carbeniumhexafluoro-phosphate (HSPyU), chlorodipyrrolidinocarbeniumhexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolidiniumtetrafluoroborate(CIB), (benzotriazol-1-yloxy) dipiperidinocarbeniumhexafluorophosphate (HBPipU),O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TCTU), bromotris(dimethylamino)-phosphoniumhexafluorophosphate (BroP), propylphosphonic anhydride (PPACA,2-morpholinoethyl isocyanide (MEI),N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate(HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP),O-[(ethoxycarbonyl)cyanomethylenamino]-N,N,N′,N′-tetramethyl-uroniumtetrafluoroborate (TOTU),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride(MMTM, DMTMM), N,N,N′,N-tetram ethyl-O—(N-succinimidyl)-uroniumtetrafluoroborate (TSTU),O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoro-borate (TDBTU), 1,1′-(azodicarbonyl)dipiperidine (ADD),di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butylazodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), diethylazodicarboxylate (DEAD).

Another aspect of the invention provides a chemical syntheticpreparation of derivatives of amanita toxins of Formula (I) which areillustrated in FIGS. 1-28. The synthetic preparations can be in solidphase, solution phase or combination of solid and solution phases.

Because the cytotoxic agents of formula (I) of the present inventioncontain asymmetrically substituted carbon atoms, and may be isolated inoptically active or racemic forms, all chiral, diastereomeric, racemicforms and all geometric isomeric forms of a structure are intended,unless the specific stereochemistry or isomeric form is specificallyindicated. It is well known in the art how to prepare and isolate suchoptically active forms. For example, mixtures of stereoisomers may beseparated by standard techniques including, but not limited to,resolution of racemic forms, normal, reverse-phase, and chiralchromatography, preferential salt formation, recrystallization, and thelike, or by chiral synthesis either from chiral starting materials or bydeliberate synthesis of target chiral centers.

The cytotoxic agents of the present invention may be prepared by avariety of synthetic routes. The reagents and starting materials arecommercially available, or readily synthesized by well-known techniquesby one of ordinary skill in the arts. All substituents, unless otherwiseindicated, are as previously defined.

In the synthetic reactions of the cytotoxic agents of the presentinvention, it may be necessary to protect reactive functional groups,for example hydroxy, amino, imino, thio or carboxy groups, where theseare desired in the final product, to avoid their unwanted participationin the reactions. Conventional protecting groups may be used inaccordance with standard practice, for examples see Peter G. M. Wuts,Theodora W. Greene in “Greene's Protective Groups in Organic Synthesis”,4th Edition, John Wiley and Sons, 2006; Ian T. Harrison, Shuyen Harrisonin “Compendium of Organic Synthetic Methods”, Vol 1, 2 Vols. 1& 2 By IanT. Harrison & Shuyen Harrison, Vols 3˜5 by Louis S. Hegedus, Leroy WadeVols 6˜Vol 12 by Michael B. Smith, John Wiley and Sons, 2006-2012.

Normally the synthetic reactions are carried out in suitable solvents,temperatures and time. A variety of solvents which have no adverseeffect on the reaction or on the reagents involved can be used in asynthetic reaction of the cytotoxic agent. Examples of suitable solventsinclude: hydrocarbons, which may be aromatic, aliphatic orcycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene,toluene and xylene; hydrocarbons containing halogens, such aschloroform, dichloromethane, dichloroethane; amides, such asdimethylactamide or dimethylformamide; alcohols such as propanol,ethanol or methanol, and ethers, such as diethyl ether, tetrahydrofuran,or dioxane. The reactions can take place over a wide range oftemperatures, from −100° C.˜300° C., preferably from 0° C. to 100° C.The time required for the synthetic reaction may also vary widely,depending on many factors, notably the reaction temperature and thenature of the reagents and can be from 5 second to 4 weeks, morepreferably from 10 min to 20 hours. In addition, the cytotoxic agentsprepared may be isolated or purified from the reaction mixture byconventional means, such as evaporating or distilling off the solventfrom the reaction mixture, or after distilling off the solvent from thereaction mixture, pouring the residue into water followed by extractionwith a water-immiscible organic solvent and then distilling off thesolvent from the extract. It may also involve various well knowntechniques, such as re-crystallization, re-precipitation or the variouschromatography techniques, notably column chromatography, preparativethin layer chromatography, or high performance liquid chromatography.

Some of the synthetic reactions of the cytotoxic agents and theirconjugates to a cell binding agent are further exampled but notrestricted in the FIGS. 1-28 and in the examples 1˜70 of thedescription,

The Conjugates of Cell-Binding Agent—Cytotoxic Agent

The present invention also provides a conjugate molecule comprising atleast one derivative of amanita toxins covalently linked to a cellbinding agent (CBA) through the linking group of the crosslinker (L).Preferably said conjugate comprises one to twenty molecules ofderivatives of amanita toxins according to the invention covalentlylinked to a cell binding agent through the linking group of the linkerof the derivatives of amanita toxins.

As stated above, the conjugates of a cell surface bindingmolecule—cytotoxic agent are illustrated in the Formula (I).

or their pharmaceutically acceptable salts, hydrates, or hydrated salts;or the polymorphic crystalline structures of these compounds; or theiroptical isomers, racemates, diastereomers or enantiomers.

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, L, m, n and Q aredescribed the same as early in Formula (I). L is preferred a linker-cellbinding molecule covalently bound cluster.

In certain embodiments, the conjugates of the invention are illustratedin the following formula:

Wherein Aa, L, m, n, p, Q, r, R₁, and R₂, are described above in Formula(I). CBA is a Cell binding agent.

Drug loading (DAR) of the conjugates may range from 1 to 20 drugmoieties (D) per cell binding agent and is preferred the average numberof 2-8 drug moieties per cell binding agent in a molecule of Formula(II-1)-(II-91). When CBA is antibody in preparations of ADC, thepreferred drug loading is 3˜6 drug per antibody and the average numberof drug moieties per antibody from conjugation reactions may becharacterized by conventional means such as mass spectroscopy (HPLC-MS,UPLC-QTOF, HPLC-MS/MS), ELISA assay, and HPLC (SEC-HPLC, HIC-HPLC). Thequantitative distribution of the conjugates in terms of the drug loadingmay also be determined. In some instances, separation, purification, andcharacterization of homogeneous the conjugates where drug loading is acertain value from the conjugates with the drug loadings may be achievedby means such as reverse phase HPLC or electrophoresis.

The Cell binding agents (CBA) may be of any kind and include peptidesand non-peptides. Generally, the cell binding agents include, but arenot limited to, large molecular weight proteins such as, for example,full-length antibodies (polyclonal and monoclonal antibodies); singlechain antibodies; fragments of antibodies such as Fab, Fab′, F(ab′)₂,F_(v), [Pharma, J. Immunol. 131, 2895-2902 (1983)], fragments producedby a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR's,and epitope-binding fragments of any of the above whichimmuno-specifically bind to cancer cell antigens, viral antigens ormicrobial antigens; antibody mimetic, such as an affibody; domainantibodies (dAb); nanobodies; unibodies; DARPins; anticalins;versabodies; duocalins; lipocalins; vimers; interferons (such as type I,II, III); peptides; lymphokines such as IL-2, IL-3, IL-4, IL-5, IL-6,GM-CSF, interferon-gamma (ITN-γ); hormones such as insulin, TRH(thyrotropin releasing hormones), MSH (melanocyte-stimulating hormone),steroid hormones, such as androgens and estrogens,melanocyte-stimulating hormone (MSH); growth factors andcolony-stimulating factors such as epidermal growth factors (EGF),granulocyte-macrophage colony-stimulating factor (GM-CSF), transforminggrowth factors (TGF), such as TGFα, TGFβ, insulin and insulin likegrowth factors (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF [Burgess,Immunology Today, 5, 155-158 (1984)]; vaccinia growth factors (VGF);fibroblast growth factors (FGFs); smaller molecular weight proteins,poly-peptide, peptides and peptide hormones, such as bombesin, gastrin,gastrin-releasing peptide; platelet-derived growth factors; interleukinand cytokines, such as interleukin-2 (IL-2), interleukin-6 (IL-6),leukemia inhibitory factors, granulocyte-macrophage colony-stimulatingfactor (GM-CSF); vitamins, such as folate; apoproteins andglycoproteins, such as transferrin {O'Keefe et al, 260 J. Biol. Chem.932-937 (1985)}; sugar-binding proteins or lipoproteins, such aslectins; cell nutrient-transport molecules; and small molecularinhibitors, such as prostate-specific membrane antigen (PSMA) inhibitorsand small molecular tyrosine kinase inhibitors (TKI), non-peptides orany other cell binding molecule or substance, such as bioactive polymers(Dhar, et al, Proc. Natl. Acad. Sci. 2008, 105, 17356-61) or a polymerhaving a cell binding agent on its surface; dendrimers (Lee, et al, Nat.Biotechnol. 2005, 23, 1517-26; Almutairi, et al; Proc. Natl. Acad. Sci.2009, 106, 685-90) or a dendrimer containing a cell binding agent;nanoparticles (Liong, et al, ACS Nano, 2008, 19, 1309-12; Medarova, etal, Nat. Med. 2007, 13, 372-7; Javier, et al, Bioconjugate Chem. 2008,19, 1309-12) or a nanoparticles having a cell binding agent on itssurface; liposomes (Medinai, et al, Curr. Phar. Des. 2004, 10, 2981-9)or a liposome having a cell binding agent; viral capsides (Flenniken, etal, Viruses Nanotechnol. 2009, 327, 71-93). In general monoclonalantibodies are preferred as a cell-surface binding agent if anappropriate one is available.

The linker used for the conjugation of this invention includes, but notlimited to, a disulfide linker, a thioether linker, an amide bondedlinker, a peptidase-labile linker, a photolabile linker, an acid-labilelinkers (such as hydrazone liner), an esterase-labile linker, anoxidatively labile linker, a metabolically labile linker, abiochemically labile linker.

Preferably, the linker is linked to the cell binding agent via afunction reactive towards for instance thiol and amino functions of thecell binding agent coming from reduced disulfide bonds and lysineresidues respectively. More particularly, said derivative is linkedthrough the —CO— group to the amino function of the lysine residue ofsaid cell binding agent, so as to form an amide bond.

In addition, the linker may be composed of one or more linkercomponents. Exemplary linker components include 6-maleimidocaproyl(“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or“vc”), alanine-phenylalanine (“ala-phe” or “af”), glycine-glycine, anature peptides containing up to 6 the same or different aminoacides(dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide),p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“STAB”), ethyleneoxy (—CH₂CH₂O—) as one orup to 100 repeating units (“EO” or “PEO”). The linker may be a“cleavable linker,” facilitating release of a drug in the cell.Additional linker components are known in the art and some areillustrated below:

Wherein R₁₀ is defined in Formula (I) above. Wherein R₁₅, R₁₆ and R₁₇are independently selected from —C₁˜C₈ alkyl or alkylene-, —C₁˜C₇carbocyclo-, alkyl)-, —NH—(C₁˜C₈ alkyl)-, -arylene-, —C₁˜C₅alkylene-arylene-, -arylene, —C₁˜C₈ alkylene-, —C₁˜C₈ alkylene-(C₁˜C₈carbocyclo)-, —(C₃˜C₇ carbocyclo)-C₁˜C₈ alkylene-, —C₃˜C₈ heterocyclo-,—C₁˜C₈ alkylene-(C₃˜C₈ heterocyclo)-, —(C₃˜C₈ heterocyclo)-C₁˜C₉alkylene-, —(CH₂CH₂O)_(k)—, —(CH(CH₃)CH₂O)_(k)—, and—(CH₂CH₂O)_(k)—CH₂—; k is an integer ranging from 1-30; X′″, Y′″ and Z′″are independently selected from NH, O or S.

In a preferred embodiment, conjugates of the invention areantibody/cytotoxic agent, antibody fragment/cytotoxic agent,diabody/cytotoxic agent, tri(a)body/cytotoxic agent, epidermal growthfactor (EGF)/cytotoxic agent, prostate specific membrane antigen (PSMA)inhibitor/cytotoxic agent, melanocyte stimulating hormone(MSH)/cytotoxic agent, thyroid stimulating hormone (TSH)/cytotoxicagent, polyclonal antibody/cytotoxic agent, somatostatin/cytotoxicagent, folate/cytotoxic agent, matriptase inhibitor/cytotoxic agent,estrogen/cytotoxic agent, estrogen analogue/cytotoxic agent, designedankyrin repeat proteins (DARPins)/cytotoxic agent, androgen/cytotoxicagent, and androgen analogue/cytotoxic agent.

In a more preferred embodiment, conjugates of the invention aremonoclonal antibody/cytotoxic agent. Examples of antibodies used forconjugation of cyotoxic agents in this prevention include, but are notlimited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab (antiCD4l (integrin alpha-IIb), Adalimumab (anti-TNF-α), Adecatumumab(anti-EpCAM, CD326), Afelimomab (anti-TNF-α); Afutuzumab (anti-CD20),Alacizumab pegol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab(Campath, MabCampath, anti-CD52), Altumomab (anti-CEA), Anatumomab(anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab(anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin(CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6receptor), Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-betaamyloid), Basiliximab (Simulect, antiCD25 (α chain of IL-2 receptor),Bavituximab (anti-phosphatidylserine), Bectumomab (LymphoScan,anti-CD22), Belimumab (Benlysta, LymphoStat-B, anti-BAFF), Benralizumab(anti-CD125), Bertilimumab (anti-CCL11 (eotaxin-1)), Besilesomab(Scintimun, anti-CEA-related antigen), Bevacizumab (Avastin,anti-VEGF-A), Biciromab (FibriScint, anti-fibrin II beta chain),Bivatuzumab (anti-CD44 v6), Blinatumomab (BiTE, anti-CD19), Brentuximab(cAC10, anti-CD30 TNFRSF8), Briakinumab (anti-IL-12, IL-23) Canakinumab(Ilaris, anti-IL-1), Cantuzumab (C242, anti-CanAg), Capromab,Catumaxomab (Removab, anti-EpCAM, anti-CD3), CC49 (anti-TAG-72),Cedelizumab (anti-CD4), Certolizumab pegol (Cimzia anti-TNF-α),Cetuximab (Erbitux, IMC-C225, anti-EGFR), Citatuzumab bogatox(anti-EpCAM), Cixutumumab (anti-IGF-1), Clenoliximab (anti-CD4),Clivatuzumab (anti-MUC1), Conatumumab (anti-TRAIL-R2), CR6261(anti-Influenza A hemagglutinin), Dacetuzumab (anti-CD40), Daclizumab(Zenapax, anti-CD25 (a chain of IL-2 receptor)), Daratumumab (anti-CD38(cyclic ADP ribose hydrolase), Denosumab (Prolia, anti-RANKL), Detumomab(anti-B-lymphoma cell), Dorlimomab, Dorlixizumab, Ecromeximab (anti-GD3ganglioside), Eculizumab (Soliris, anti-C5), Edobacomab(anti-endotoxin), Edrecolomab (Panorex, MAb17-1A, anti-EpCAM),Efalizumab (Raptiva, anti-LFA-1 (CD11a), Efungumab (Mycograb,anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab (anti-IL-6),Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab (anti-episialin),Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab(Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin, anti-integrinα_(v)β₃), Exbivirumab (anti-hepatitis B surface antigen), Fanolesomab(NeutroSpec, anti-CD15), Faralimomab (anti-interferon receptor),Farletuzumab (anti-folate receptor 1), Felvizumab (anti-respiratorysyncytial virus), Fezakinumab (anti-IL-22), Figitumumab (anti-IGF-1receptor), Fontolizumab (anti-IFN-γ), Foravirumab (anti-rabies virusglycoprotein), Fresolimumab (anti-TGF-β), Galiximab (anti-CD80),Gantenerumab (anti-beta amyloid), Gavilimomab (anti-CD147 (basigin)),Gemtuzumab (anti-CD33), Girentuximab (anti-carbonic anhydrase 9),Glembatumumab (CR011, anti-GPNMB), Golimumab (Simponi, anti-TNF-α),Gomiliximab (anti-CD23 (IgE receptor)), Ibalizumab (anti-CD4),Ibritumomab (anti-CD20), Igovomab (Indimacis-125, anti-CA-125),Imciromab (Myoscint, anti-cardiac myosin), Inflixinnab (Remicade,anti-TNF-α), Intetumumab (anti-CD51), Inolimomab (anti-CD25 (a chain ofIL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152),Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4), Labetuzumab(CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13), Lemalesomab(anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2),Lexatumumab (anti-TRAIL-R₂), Libivirumab (anti-hepatitis B surfaceantigen), Lintuzumab (anti-CD33), Lucatumumab (anti-CD40), Lumiliximab(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria,anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside),Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratorysyncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab(anti-C₂₄₂), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrinα4), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab(anti-TNF-α), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab,Ocrelizumab (anti-CD20), Odulimomab (Afolimomab, anti-LFA-1 (CD11a)),Ofatumumab (Arzerra, anti-CD20), Olaratumab (anti-PDGF-Rα), Omalizumab(Xolair, anti-IgE Fc region), Oportuzumab (anti-EpCAM), Oregovomab(OvaRex, anti-CA-125), Otelixizumab (anti-CD3), Pagibaximab(anti-lipoteichoic acid), Palivizumab (Synagis, Abbosynagis,anti-respiratory syncytial virus), Panitumumab (Vectibix, ABX-EGF,anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa), Pascolizumab(anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab (Omnitarg,2C4, anti-HER2/neu), Pexelizumab (anti-C5), Pintumomab(anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab(anti-vimentin), PRO 140 (anti-CCRS), Racotumomab (1E10,anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)),Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2),Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin,protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B),Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera,Rituxanmab, anti-CD20), Robatumumab (anti-IGF-1 receptor), RontalizumabRovelizumab (LeukArrest, anti-CD11, CD18), Ruplizumab (Antova,anti-CD154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab(anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab(anti-IFN-α), Siltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart)MI95 (anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab(anti-sphingosine-1-phosphate), Sontuzumab (anti-episialin), Staniulumab(anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyteantigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab(anti-integrin α_(11b)β₃), Talizumab (anti-IgE), Tanezumab (anti-NGF),Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factorA), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40),Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,(anti-CTLA-4), Tigatuzumab (anti-TRAIL-R₂), TNX-650 (anti-IL-13),Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor),Toralizumab (anti-CD154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab(Herceptin, (anti-HER2/neu), Tremelimumab (anti-CTLA-4), Tucotuzumabcelmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus),Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12,IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab, (anti-integrinα₄β₇), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1),Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),Volociximab (anti-integrin α₅β₁), Votumumab (HumaSPECT, anti-tumorantigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR), Zanolimumab(HuMax-CD4, anti-CD4), Ziralimumab (anti-CD147 (basigin)), Zolimomab(anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept(Orencia®), Rilonacept (Arcalyst), 14F7 [anti-IRP-2 (Iron RegulatoryProtein 2)], 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst. formelanoma and solid tumors), J591 (anti-PSMA, Weill Cornell MedicalSchool for prostate cancers), 225.28S [anti-HMW-MAA (High molecularweight-melanoma-associated antigen), Sorin Radiofarmaci S. R. L. (Milan,Italy) for melanoma], COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst.USA for colorectal and gastric cancers), CYT-356 (Oncoltad®, forprostate cancers), HNK20 (OraVax Inc. for respiratory syncytial virus),ImmuRAIT (from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, PeregrinePharm. for Cancers), MAK-195F [anti-TNF (tumor necrosis factor; TNFA,TNF-alpha; TNFSF2), from Abbott/Knoll for Sepsis toxic shock], MEDI-500[T10B9, anti-CD3, TRαβ (T cell receptor alpha/beta), complex, fromMedImmune Inc for Graft-versus-host disease], RING SCAN [anti-TAG 72(tumour associated glycoprotein 72), from Neoprobe Corp. for Breast,Colon and Rectal cancers], Avicidin (anti-EPCAM (epithelial celladhesion molecule), anti-TACSTD1 (Tumor-associated calcium signaltransducer 1), anti-GA733-2 (gastrointestinal tumor-associated protein2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA; KS1/4 antigen;M4S; tumor antigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian,Prostate cancers and NHL]; LymphoCide (Immunomedics, NJ), Smart IDIO(Protein Design Labs), Oncolym (Techniclone Inc, CA), Allomune(BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics,NJ), IMC-1C11 (ImClone, NJ) and Cetuximab (ImClone, NJ).

Other antibodies as binding ligands include, but are not limited to, areantibodies against the following antigens: Aminopeptidase N (CD13),Annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian), CA15-3(carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y(carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas),CA242 (colorectal), placental alkaline phosphatase (carcinomas),prostate specific antigen (prostate), prostatic acid phosphatase(prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin'sdisease, NHL lymphoma, multiple myeloma), CD3 epsilon (T cell lymphoma,lung, breast, gastric, ovarian cancers, autoimmune diseases, malignantascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma),CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin'slymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiplemyeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51(Metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lungcancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor,multiple myeloma), CD66e (cancers), CD70 (metastatic renal cellcarcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80(lymphoma), CD98 (cancers), mucin (carcinomas), CD221 (solid tumors),CD227 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309(ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastriccancers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (breast,colorectal and lung cancers), DLL4 (A-like-4), EGFR (Epidermal GrowthFactor Receptor, various cancers), CTLA4 (melanoma), CXCR4 (CD184,Heme-oncology, solid tumors), Endoglin (CD105, solid tumors), EPCAM(epithelial cell adhesion molecule, bladder, head, neck, colon, NHLprostate, and ovarian cancers), ERBB2 (Epidermal Growth Factor Receptor2; lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR(folate receptor, ovarian cancers), GD2 ganglioside (cancers), G-28 (acell surface antigen glyvolipid, melanoma), GD3 idiotype (cancers), Heatshock proteins (cancers), HER1 (lung, stomach cancers), HER2 (breast,lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cellleukemia), human chorionic gonadotropin (carcinoma), IGF1R (insulin-likegrowth factor 1 receptor, solid tumors, blood cancers), IL-2 receptor(interleukin 2 receptor, T-cell leukemia and lymphomas), IL-6R(interleukin 6 receptor, multiple myeloma, RA, Castleman's disease, IL6dependent tumors), Integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, forvarious cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3(carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor(carcinomas), p 97 (melanoma), MS4A1 (membrane-spanning 4-domainssubfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 orMUC1-KLH (breast, ovarian, cervix, bronchus and gastrointestinalcancer), MUC16 (CA125) (Ovarian cancers), CEA (colorectal), gp100(melanoma), MART1 (melanoma), MPG (melanoma), MS4A1 (membrane-spanning4-domains subfamily A, small cell lung cancers, NHL), Nucleolin, Neuoncogene product (carcinomas), P21 (carcinomas), Paratope ofanti-N-glycolylneuraminic acid (Breast, Melanoma cancers), PLAP-liketesticular alkaline phosphatase (ovarian, testicular cancers), PSMA(prostate tumors), PSA (prostate), ROBO4, TAG 72 (tumour associatedglycoprotein 72, AML, gastric, colorectal, ovarian cancers), T celltransmembrane protein (cancers), Tie (CD202b), TNFRSF10B (tumor necrosisfactor receptor superfamily member 10B, cancers), TNFRSF13B (tumornecrosis factor receptor superfamily member 13B, multiple myeloma, NHL,other cancers, RA and SLE), TPBG (trophoblast glycoprotein, Renal cellcarcinoma), TRAIL-R₁ (Tumor necrosis apoprosis Inducing ligand Receptor1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma),VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumorassociated antigens recognized by antibodies have been reviewed (Gerber,et al, mAbs 1:3, 247-253 (2009); Novellino et al, Cancer ImmunolImmunother. 54(3), 187-207 (2005). Franke, et al, Cancer BiotherRadiopharm. 2000, 15, 459-76). Examples of these antigens thatantibodies against are: Many other Cluster of Differentiations (CD4,CDS, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15,CD16, CDw17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD34, CD35, CD36, CD37, CD38, CD41, CD42, CD43,CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD56,CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD70, CD71,CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD9O, CD91, CD95,CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD127,CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151,CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186, CD195,CD202 (a, b), CD209, CD235a, CD271, CD303, CD304), Annexin A1,Nucleolin, Endoglin (CD105), ROBO4, Amino-peptidase N, A-like-4 (DLL4),VEGFR-2 (CD309), CXCR4 9CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7,EGFRvIII, HER-2/neu, Idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, GD2,CEA, MelanA/MARTI, Ras mutant, gp100, p53 mutant, Proteinase3 (PR1),bcr-abl, Tyrosinase, Survivin, hTERT, Sarcoma translocation breakpoints,EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17,PAX3, ALK, Androgen receptor, Cyclin B1, Polysialic acid, MYCN, RhoC,TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE A1, sLe(a), CYP1B1,PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn,Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA,AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1,FAP, PDGFR-β, MAD-CT-2, Fos-related antigen 1.

Production of antibodies used in the present invention involves in vivoor in vitro procedures or combinations thereof. Methods for producingpolyclonal anti-receptor peptide antibodies are well-known in the art,such as in U.S. Pat. No. 4,493,795 (to Nestor et al). A monoclonalantibody is typically made by fusing myeloma cells with the spleen cellsfrom a mouse that has been immunized with the desired antigen (Kohler,G.; Milstein, C. (1975). Nature 256: 495-497). The detailed proceduresare described in “Antibodies—A Laboratory Manual”, Harlow and Lane,eds., Cold Spring Harbor Laboratory Press, New York (1988), which isincorporated herein by reference. Particularly monoclonal antibodies areproduced by immunizing mice, rats, hamsters or any other mammal with theantigen of interest such as the intact target cell, antigens isolatedfrom the target cell, whole virus, attenuated whole virus, and viralproteins. Splenocytes are typically fused with myeloma cells usingpolyethylene glycol (PEG) 6000. Fused hybrids are selected by theirsensitivity to HAT (hypoxanthine-aminopterin-thymine). Hybridomasproducing a monoclonal antibody useful in practicing this invention areidentified by their ability to immunoreact specified receptors orinhibit receptor activity on target cells.

A monoclonal antibody used in the present invention can be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques, such as using protein-A, or protein-G affinitychromatography; anion, cation, hydrophobic, or size exclusivechromatographies (particularly by affinity for the specific antigenafter Protein A or G, and sizing column chromatography); centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media. An exemplary synthetic medium is Dulbecco's minimalessential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959))supplemented with 4.5 gm/l glucose, 20 mm glutamine, 20% fetal calfserum and with an anti-foaming agent, such aspolyoxyethylene-polyoxypropylene block copolymer.

In addition, antibody-producing cell lines can also be created bytechniques other than cell fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with an oncovirus, suchas Epstein-Barr virus (EBV, also called human herpesvirus 4 (HHV-4)) orKaposi's sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890. A monoclonal antibody may also beproduced via an anti-receptor peptide or peptides containing thecarboxyl terminal as described well-known in the art. See Niman et al.,Proc. Natl. Acad. Sci. USA, 80: 4949-4953 (1983); Geysen et al., Proc.Natl. Acad. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry34(20): 6675-6688, (1995). Typically, the anti-receptor peptide or apeptide analog is used either alone or conjugated to an immunogeniccarrier, as the immunogen for producing anti-receptor peptide monoclonalantibodies.

There are also a number of other well-known techniques for makingmonoclonal antibodies as binding molecules in this invention.Particularly useful are methods of making fully human antibodies. Onemethod is phage display technology which can be used to select a rangeof human antibodies binding specifically to the antigen using methods ofaffinity enrichment. Phage display has been thoroughly described in theliterature and the construction and screening of phage display librariesare well known in the art, see, e.g., Dente et al, Gene. 148(1):7-13(1994); Little et al, Biotechnol Adv. 12(3):539-55 (1994); Clackson etal., Nature 352:264-268 (1991); Huse et al., Science 246:1275-1281(1989), Hoogenboom et al. in Methods in Molecular Biology 178:1-37(2001) (O'Brien et al., ed., Human Press, Totowa, N.J.), and in certainembodiments, in Lee et al. J. Mol. Biol. 340:1073-1093 (2004).

Moncolonal antibodies derived by hybridoma technique from anotherspecies than human, such as mouse, can be humanized to avoid humananti-mouse antibodies when infused into humans. Among the more commonmethods of humanization of antibodies are complementarity-determiningregion grafting and resurfacing. These methods have been extensivelydescribed, see e.g. U.S. Pat. Nos. 5,859,205 and 6,797,492; Liu et al,Immunol Rev. 222:9-27 (2008); Almagro et al, Front Biosci. 1; 13:1619-33(2008); Lazar et al, Mol Immunol. 44(8):1986-98 (2007); Li et al, Proc.Natl. Acad. Sci. USA. 103(10):3557-62 (2006) each incorporated herein byreference. Fully human antibodies can also be prepared by immunizingtransgenic mice, rabbits, monkeys, or other mammals, carrying largeportions of the human immunoglobulin heavy and light chains, with animmunogen. Examples of such mice are: the Xenomouse (Abgenix/Amgen.),the HuM Ab-Mouse (Medarex/BMS), the VelociMouse (Regeneron), see alsoU.S. Pat. Nos. 6,596,541, 6,207,418, 6,150,584, 6,111,166, 6,075,181,5,922,545, 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In humantherapy, murine variable regions and human constant regions can also befused to construct called “chimeric antibodies” that are considerablyless immunogenic in man than murine mAbs (Kipriyanov et al, MolBiotechnol. 26: 39-60 (2004); Houdebine, Curr Opin Biotechnol. 13: 625-9(2002) each incorporated herein by reference). In addition,site-directed mutagenesis in the variable region of an antibody canresult in an antibody with higher affinity and specificity for itsantigen (Brannigan et al, Nat Rev Mol Cell Biol. 3: 964-70, (2002));Adams et al, J Immunol Methods. 231: 249-60 (1999)) and exchangingconstant regions of a mAb can improve its ability to mediate effectorfunctions of binding and cytotoxicity.

Antibodies immunospecific for a malignant cell antigen can also beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., chemical synthesis or recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a malignant cell antigen can be obtained commercially, e.g., fromthe GenBank database or a database like it, the literature publications,or by routine cloning and sequencing.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the antibody can be readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells (Skerra et al.,Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,130: 151 (1992)). Antibodies can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies. Such modulation is accomplished atleast in part by simultaneously modulating translational strengths forthe polypeptide components. After the fermentation which is known in theart, the produced antibody protein is further purified to obtainpreparations that are substantially homogeneous for further assays anduses. Standard protein purification methods known in the art can beemployed. The exemplary purification procedures: fractionation onimmunoaffinity (such as Protein A columns) or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

Apart from an antibody, a peptide or protein that bind/block/target orin some other way interact with the epitopes or corresponding receptorson a targeted cell can be used as a binding molecule. These peptides orproteins could be any random peptide or proteins that have an affinityfor the epitopes or corresponding receptors and they don't necessarilyhave to be of the immunoglobulin family. These peptides can be isolatedby similar techniques as for phage display antibodies (Szardenings, JRecept Signal Transduct Res. 23(4): 307-49, 2003). The use of peptidesfrom such random peptide libraries can be similar to antibodies andantibody fragments. The binding molecules of peptides or proteins may beconjugated on or linked to a large molecules or materials, such as, butis not limited, an albumin, a polymer, a liposome, a nano particle, aslong as such attachment permits the peptide or protein to retain itsantigen binding specificity.

Any one of several different reactive groups on a cell binding agent,preferably on an antibody, can be a conjugation site, such as c-aminogroups in lysine residues, pendant carbohydrate moieties, carboxylicacid groups, disulfide groups, and thiol groups. For reviews on antibodyreactive groups suitable for conjugation, see, e.g., Hermanson, G. T.(2008). Bioconjugate Techniques, Academic Press; Garnett, Adv. DrugDelivery Rev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology& Therapeutics 83 (1999), 67-123, the disclosures of which areincorporated herein by reference.

The cytotoxic agents of this invention can be directly conjugated(linked) to a cell binding agent, or via a bifunctional linker or acrosslinking agent to a cell binding agent. The bifunctional linkerpossess two reactive groups; one of which is capable of reacting with acell binding agent while the other one reacts with one or more moleculesof cytotoxic agent of the invention. The bifunctional crosslinkers arewell known in the art (see, for example, U.S. Pat. No. 5,208,020; Isalmand Dent in “Bioconjugation” chapter 5, p 218-363, Groves DictionariesInc. New York, 1999). Examples of bifunctional linker are:N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB),N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),N-succinimidyl-3-(2-pyridyldithio)-butyrate (SDPB), 2-iminothiolane,N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB),N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SNPP),N-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SSNPB),N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SMNP),N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate (SSNPP),4-succinimidyl-oxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT),N-sulfosuccinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate(SSMNP); N-succinimidyl-4-methyl-4-(2-pyridyldithio)pentanoate (SMPDP),N-succinimidyl-4-(5-N,N-dimethyl-carboxamido-2-pyridyldithio) butyrate(SCPB),N-sulfosuccinimidyl-4-(5-N,N-dimethyl-carboxamido-2-pyridyldithio)butyrate (SSCPB),N-succinimidyl-4,4-dimethyl-4-(2-pyridyldithio)pentanoate (SDMPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB),bis-maleimidopolyethyleneglycol (BMPEG), BM(PEG)_(1˜20),N-(β-maleimidopropyloxy)-succinimide ester (BMPS), iminothiolane (IT),dimethyl adipimidate HCl or derivatives of imidoesters, active esters(such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde),bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine),bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene), gamma-maleimidobutyric acidN-succinimidyl ester (GMBS), E-maleimidocaproic acidN-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS,N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate)(a “long chain” analog of SMCC (LC-SMCC)),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH),N-succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyliodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester(KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB),succinimidyl-6-(beta-maleimidopropionamido)-hexanoate (SMPH),succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane(DTME), 1,4-bis-maleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane(BMOE), sulfosuccinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (sulfo-SMCC),sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS),N-(gamma-maleimidobutryloxy)sulfo-succinimdeester (sulfo-GMBS),N-(epsilon-maleimidocaproyloxy)sulfosuccimido ester (sulfo-EMCS),N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB); or thecommercially available linkers (such as from Thermo Scientific's Pierce:Imidoester Crosslinkers: DMA (Dimethyl adipimidate.2 HCl), DMP (Dimethylpimelimidate.2 HCl), DMS (Dimethyl Suberimidate.2 HCl), DTBP (Dimethyl3,3′-dithiobispropionimidate.2 HCl), NHS-ester Crosslinkers-AmineReactive: BS(PEG)₅ (Bis(succinimidyl) penta(ethylence glycol), BS(PEG)₉(Bis(succinimidyl) nona(ethylence glycol), BS³ (Bis[sulfosuccinimidyl]suberate), BSOCOES (Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DSG(Disuccinimidyl glutarate), DSP (Dithiobis[succinimidyl propionate]),DSS (Disuccinimidyl suberate), DST (Disuccinimidyl tartarate), DTSSP(3,3′-Dithiobis[sulfosuccinimidylpropionate]), EGS (Ethylene glycolbis[succinimidylsuccinate]), Sulfo-EGS (Ethylene glycolbis[sulfosuccinimidylsuccinate]), TSAT (Tris-succinimidylaminotriacetate), DFDNB (1,5-Difluoro-2,4-dinitrobenzene);Amine-to-Sulfhydryl Crosslinkers: Sulfo-SIAB (Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate), SLAB (Succinimidyl(4-iodoacetyl)aminobenzoate), SBAP(Succinimidyl3-(bromoacetamido)propionate), SIA (Succinimidyl iodoacetate),Sulfo-SMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate),SM(PEG)n (NHS-PEG-Maleimide Crosslinkers:Succinimidyl-([N-maleimidopropionamido])-#ethyleneglycol)ester, #=1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24),LC-SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), Sulfo-EMCS(N-epsilon-Maleimidocaproyl-oxysulfosuccinimide ester), EMCS(N-epsilon-Malemidocaproyl-oxysuccinimide ester), Sulfo-GMBS(N-gamma-Maleimidobutyryl-oxysulfosuccinimide ester), GMBS(N-gamma-Maleimidobutyryl-oxysuccinimide ester), Sulfo-KMUS(N-kappa-Maleimidoundecanoyl-oxysulfosuccinimide ester), Sulfo-MBS(m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), MBS(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-SMPB((Sulfosuccinimidyl 4-(p-maleii midophenyl)butyrate), SMPB (Succinimidyl4-(p-maleimidophenyl)butyrate), AMAS N-(a-Maleimidoacetoxy) succinimideester), BMPS (N-beta-Maleimidopropyl-oxysuccinimide ester), SMPH(Succinimidyl 6-[(beta-maleimidopropionamido)hexanoate]), PEG12-SPDP(2-Pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide),PEG4-SPDP (2-Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide),Sulfo-LC-SPDP (Sulfosuccinimidyl6-[3′-(2-pyridyldithio)propionamido]hexanoate), LC-SPDP (Succinimidyl6-[3-(2-pyridyldithio)propionamido]hexanoate), SMPT(4-Succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene);Carboxyl-to-Amine Crosslinkers: DCC (Dicyclohexylcarbodiimide), EDC(1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide); PhotoreactiveCrosslinkers: ANB-NOS (N-5-Azido-2-nitrobenzoyloxysuccinimide),NHS-Diazirine (SDA) Crosslinkers: SDA (NHS-Diazirine) (Succinimidyl4,4′-azipentanoate), LC-SDA (NHS-LC-Diazirine) (Succinimidyl6-(4,4′-azipentanamido)hexanoate), SDAD (NHS-SS-Diazirine) (Succinimidyl2-([4,4′-azipentanamido]ethyl)-1,3′-dithiopropionate), Sulfo-SDA(Sulfo-NHS-Diazirine) (Sulfosuccinimidyl 4,4′-azipentanoate),Sulfo-LC-SDA (Sulfo-NHS-LC-Diazirine) (Sulfosuccinimidyl6-(4,4′-azipentanamido)hexanoate), Sulfo-SDAD (Sulfo-NHS-SS-Diazifine)(Sulfosuccinimidyl2-([4,4′-azipentanamido]ethyl)-1,3′-dithiopropionate), Sulfo-SANPAH(Sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)-hexanoate), SPB(Succinimidyl-[4-(psoralen-8-yloxy)]-butyrate);Sulfhydryl-to-Carbohydrate Crosslinkers: BMPH (N-beta-Maleimidopropionic acid hydrazide-TFA), EMCH (N-epsilon-Maleimidocaproicacid hydrazide-TFA), KMUH (N-kappa-Maleimidoundecanoic acidhydrazide-TFA), MPBH (4-(4-N-Maleimidophenyl)butyric acidhydrazide-HCl), PDPH (3-(2-Pyridyldithio)propionyl hydrazide);Sulfhydryl-to-Hydroxyl Crosslinkers: PMPI (p-Maleimidophenylisocyanate); Sulfhydryl-to-Sulfhydryl Crosslinkers: BM(PEG)₂(1,8-Bismaleimido-diethyleneglycol), BM(PEG)3(1,11-Bismaleimido-triethyleneglycol), BMB (1,4-Bismaleimidobutane),BMDB (1,4-Bismaleimidyl-2,3-dihydroxybutane), BMH (Bismaleimidohexane),BMOE (Bismaleimidoethane), DTME (Dithiobismaleimido-ethane), TMEA(Ths(2-maleimidoethyl)amine) and SVSB(succinimidyl-(4-vinylsulfone)benzoate).

The bis-maleimide or bis-2-pyridyldithiol reagents allow the attachmentof the thiol group of a thiol-containing cell binding agent (such asantibody) to a thiol-containing drug moiety, label, or linkerintermediate, in a sequential or concurrent fashion. Other functionalgroups besides maleimide and pyridyldithiol, which are reactive with athiol group of a cell binding agent, drug moiety, label, or linkerintermediate include iodoacetamide, bromoacetamide, vinyl pyridine,disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

In additional embodiments, the linker may be composed of one or morelinker components. The exemplary linker components are:

1. The self-immolative linker components:

wherein the (*) atom is the point of attachment of additional spacer orreleasable linker units, or the cytotoxic agent, and/or the bindingmolecule (CBA); X¹, Y¹, Z² and Z³ are independently NH, or O, or S; Z¹is H, or NH, or O or S independently. v is 0 or 1; Q¹ is independentlyH, OH, C₁˜C₆ alkyl, (OCH₂CH₂)_(n)F, Cl, Br, I, OR₅, or SR₅, NR₅R₅′,N═NR₅, N═R₅NR₅R_(5′), NO₂, SOR₅R₅′, SO₂R₅, SO₃R₅, OSO₃R₅, PR₅R_(5′),POR₅R_(5′), PO₂R₅R₅′, OPO(OR₅)(OR_(5′)), or OCH₂PO(OR₅(OR_(5′)) whereinR₅ and R_(5′) are described in the Formula (I), preferably R₅ and R_(5′)are independently selected from H, C₁˜C₈ of alkyl; C₂˜C₈ of alkenyl,alkynyl, heteroalkyl; C₃˜C₈ of aryl, heterocyclic, carbocyclic,cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl; orpharmaceutical cation salts

2. The examples of non-self-immolative linker components:

Wherein the (*) atom is the point of attachment of additional spacer orreleaseable linkers, the cytotoxic agents, and/or the binding molecules;X¹, Y¹, Q¹, R₅, R_(5′) and r are as defined in Formula (I), m, n and pare 0˜6.

3. Exemplary linker components may include 6-maleimidocaproyl (“MC”),maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”),alanine-phenylalanine (“ala-phe” or “af”), p-aminobenzyloxy-carbonyl(“PAB”), N-succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”),N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”),N-Succinimidyl (4-iodo-acetyl)amino-benzoate (“SIAB”), ethyleneoxy(—CH₂CH₂O—) as one or more repeating units (“EO” or “PEO”). Additionallinker components are known in the art and some are described throughthis patent application.

In additional embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (VC or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

In the cell-binding agent—drug conjugates of the invention, cell-bindingagent (CBA) is conjugated to one or more drug moieties (Drug, or PBDderivatives), e.g. about 1 to about 20 drug moieties per cell-bindingagent, through a bifunctional linker (L). The conjugate of Formula(II-1)-(II-91) may be prepared by several routes, employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) the first modification of cell-binding agent(CBA) with a crosslinker (L) in an aqueous buffer pH 3˜9 havingoptionally 0˜30% organic co-solvents to introduce reactive disulfide,maleimido, haloacetyl, hydrazide, nitrile, alkynyl, alkyloxyamino oraldehyde groups on the cell-binding agent, to form a covalent bondedCBA-L. The CBA-L molecule then reacts with a drug moiety (Drug) offormula (I) to generate a cell binding agent—drug conjugate; or (2) thefirst modification of drug moiety (Drug) of the formula (I) with acrosslinker (L) in organic media or in an aqueous buffer pH 3˜9 havingoptionally 0˜99% organic co-solvents to introduce a reactive disulfide,maleimido, haloacetyl, hydrazide, nitrile, alkynyl, alkyloxyamino,aldehyde, N-hydroxysuccinimide (NHS) or pentafluorophenyl ester group onthe drug moiety (a covalent bonded Drug-L molecule). The Drug-L moleculethen reacts with a cell binding agent (CBA), or pre-modified CBA togenerate a cell binding agent—drug conjugate; or (3) directly throughreaction of a cell—binding agent with drug moieties of formula (I)bearing reactive function groups of disulfide, maleimido, haloacetyl,hydrazide, nitrile, alkynyl, alkyloxyamino, aldehyde,N-hydroxysuccinimide (NHS) or pentafluorophenyl esters in an aqueousbuffer pH 3˜9 having optionally 0˜30% organic co-solvents.

The thiol or amine groups on a cell-binding agents, such as an antibody,are nucleophilic and capable of reacting to form covalent bonds withelectrophilic groups on linker reagents and drug-linker intermediatesincluding: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides, such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups; and (iv) disulfides, including pyridyl disulfides, via sulfideexchange. Nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents.

Nucleophilic groups on antibodies or proteins can react to electrophilicgroups on a function linker following by reaction with a cytotoxicagent, or directly react to a linker-cytotoxic agent moiety to formcovalent bond conjugate of a cell binding agent-cytotoxic agent.Nucleophilic groups on antibodies or proteins include, but are notlimited to: (i) N-terminal amine groups, (ii) side chain amine groups,e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv)sugar hydroxyl or amino groups where the antibody is glycosylated.Amine, thiol, and hydroxyl groups are nucleophilic and capable ofreacting to form covalent bonds with electrophilic groups on linkermoieties and linker-cytotoxic agent moieties including: (i) activeesters such as NHS esters, HOBt esters, haloformates, and acid halides;(ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes,ketones, carboxyl, and maleimide groups. Certain antibodies havereducible interchain disulfides, i.e. cysteine bridges which may be madereactive by treatment with a reducing agent such as DTT(dithiothreitol), L-glutathione (GSH), dithioerythritol (DTE),2-mercaptoethylamine (β-MEA), beta mercaptoethanol (β-ME, 2-ME), ortricarbonylethylphosphine (TCEP) (Getz et al (1999) Anal. Biochem. Vol273:73-80; Soltec Ventures, Beverly, Mass.). Each cysteine bridge willthus form, theoretically, two reactive thiol nucleophiles.Alternatively, sulfhydryl groups can be introduced into antibodiesthrough modification of lysine residues, e.g., by reacting lysineresidues with 2-iminothiolane (Tract's reagent), resulting in conversionof an amine into a thiol. Reactive thiol groups may be introduced intoan antibody by introducing one, two, three, four, or more cysteineresidues (e.g., by preparing variant antibodies comprising one or morenon-native cysteine amino acid residues). Thus free thiol on the cellbinding agents can be conjugated to the thiol-reactive groups, such as,a maleimide, an iodoacetamide, a pyridyl disulfide, or otherthiol-reactive groups on the cytotoxic agents, or linker-cytotoxic agentintermediates of the invention. Some unconjugated free thiols on theantibodies can be reoxidized to reform interchain and intrachaindisulfide bonds.

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Examples of the general conjugations which a linker reacts to a drug toform a linker-drug intermediate first, following by conjugation reactionwith a cell-binding molecule are depicted below:

wherein E includes, but is not limited to, such as hydroxysuccinimidylesters (NHS, Sulfo-NHS, etc), 4-nitrophenyl esters, pentafluorophenylesters, tetrafluorophenyl (includes sulfo-tetrafluorophenyl) esters,anhydrides, acid chlorides, sulfonyl chlorides, isocyanates andisothiocyanates. R′ and R″ are independently H or CH₃, or C₂H₅; J is F,Cl, Br, I, tosylate (TsO), mesylate (MsO), nitrophenol, dinitrophenol,or pentafluorophenol; wherein Drug is a compound of the Formula (I),(Ia), (Ib), (Ic) or (Id) of this invention.

It is to be understood that where more than one nucleophilic group onthe cell binding agents, such as an antibody, reacts with a drug-linkerintermediate or linker reagent followed by drug moiety reagent, then theresulting product is a mixture of the cell binding agent-cytoxic agentconjugates with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual conjugate molecules maybe identified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography. In certain embodiments, ahomogeneous conjugate with a single loading value may be isolated fromthe conjugation mixture by electrophoresis or chromatography.

In yet another embodiment, when an IgG antibody conjugated with one, ortwo, or more the same or different of derivatives of amanita toxins ofFormula (I), (Ia), (Ib), (Ic) or (Id), optionally together with otherdifferent function molecules or cytotoxic agents, through a pair ofthiols on the IgG antibody, as exampled in formula (II-11), (II-12),(II-13), (II-14), (II-17), (II-18), (II-19), (II-20), (II-24), (II-25),(II-29), (II-30), (II-32), (II-33), (II-34), (II-35), (H-36), (II-37),(II-38), (II-39), (II-40), (II-41), (II-42), (II-43), (II-44), (II-45),(II-46), (II-47), (II-48), (II-49), (II-50), (II-51), (II-52), (II-53),and (II-64), the ADCs containing an amanita toxin of Formula (I), (Ia),(Ib), (Ic) or (Id) are preferred to be formed via a bridge linkerspecifically at the pair of thiols (through reduction of the disulfidebonds) between the light chain and heavy chain, or/and the upperdisulfide bonds between the two heavy chains, or/and the lower disulfidebonds between the two heavy chains. The ADCs containing one, or two, ormore the same or different of derivatives of amanita toxins of Formula(I), (Ia), (Ib), (Ic) or (Id) with a bridge linker are preferrablyhaving the formula of (III-1), (III-2), (III-3), (III-4), (III-5),(III-6), (III-7), (III-8), (III-9), (III-10), (III-11), or (III-12)illustrated in FIGS. 31A to 31F.

In formulae of (III-1), (III-2), (III-3), (III-4), (III-5), (III-6),(III-7), (III-8), (III-9), (III-10), (III-11), and (III-12), the IgGantibody is, preferably IgG1, IgG2, IgG3 or IgG4 antibody; “---=”represents either a single bond or a double bond; L₁ and L₂ are, thesame or deferent, independently defined the same as L in Formula (I); X₁and X₂, are, the same or deferent, independently selected from NH,N(R₁), O, S, CH₂, or Ar, wherein R₁ is C₁-C₆ alkyl, Ar is aromatic orheteroaromatic ring; wherein Drug₁ and Drug₂ are the same or deferent, aderivative of amanita toxins of Formula (I), (Ia), (Ib), (Ic) or (Id).In addition, either Drug₁ or Drug₂ can be absent, but not be absent atthe same time. When any single one of Drug₁ or Drug₂ is a derivative ofAmanita toxin of Formula (I), (Ia), (Ib), (Ic) or (Id), the other one ofeither Drug₁ or Drug₂ can be selected from (OCH₂CH₂)_(r)OR₁₀, or(OCH₂CH(CH₃))_(p)OR₁₀, or NH(CH₂CH₂O)_(F)R₁₀, or NH(CH₂CH(CH₃)O)_(p)R₁₀,or N[(CH₂CH₂O)_(p)R₁₀][(CH₂CH₂O)_(r)R₅], or (OCH₂CH₂)_(p)COOR₁₀, orCH₂CH₂(OCH₂CH₂)_(p)COOR₁₀, wherein p, r, R₁₀ are described in manyembodiments throughout this patent application. In further addition,Drug₂ and L₂ can be absent, thus X₂ is NH₂ or OH.

In yet another embodiment, when an IgG antibody conjugated with onederivative of amanita toxins of Formula (I), (Ia), (Ib), (Ic) or (Id)through a pair of thiols of an IgG antibody, the ADCs containing anamanita toxin of Formula (I), (Ia), (Ib), (Ic) or (Id) are alsopreferred to be formed via a bridge linker specifically at the pair ofthiols of IgG antibody (through reduction of the disulfide bonds)between the light chain and heavy chain, the upper disulfide bondsbetween the two heavy chains, and the lower disulfide bonds between thetwo heavy chains. The bridge linkage is preferred having the followingstructures of (IV-1), (IV-2), (IV-3), (IV-4), (IV-5), and (IV-6):

wherein“═” represents either a single bond or a double bond; Drug is aderivative of Amanita toxin of Formula (I), (Ia), (Ib), (Ic) or (Id);

represents the sites on an antibody; L, X, n and R₁₂ are defined thesame as in Formula (I).

The pairs of thiols on the antibody are preferred to be reduced by areducing agent selected from dithiothreitol (DTT), dithioerythritol(DTE), L-glutathione (GSH), tris (2-carboxyethyl) phosphine (TCEP),2-mercaptoethylamine (β-MEA), or/and beta mercaptoethanol (β-ME, 2-ME).More preferably, these reducing agents are loaded or covalently bondedto a solid polymer or a solid particle. The polymer or the particle canbe polyethene, polyacrylate, silican, crossed-linked silica (e.g.2-mercaptoethyl)silica, (aminoethyl)silica, (aminopropyl)silica),polyethylene terephthalate, polyethylene glycol, polystyrene,poly(isopropyl acrylate), dextrans (e.g. sephadex, cross-linkeddextran), isopropylacrylamide butyl methacrylate copolymer,polysaccharide polymer (e.g. agarose, agar, agaropectin, sepharose).Under the reduction of the polymer bonded reducing agents, the pairs ofdisulfide bonds can be selectively reduced at a certain position, forinstance, at the hinge region of IgG1, IgG2, IgG3 or IgG4 antibody, ordisulfide bonds between heavy chain and the light chain of the IgG1,IgG2, IgG3 or IgG4 antibody, or the certain outer surfaces of a proteinor a cell binding molecule. Thus the loading ratios as well as thepostions of derivatives of amanita toxins conjugated on the cell bindingmolecules are controlled.

In addition, when any single one of either Drug₁ or Drug₂ of Formula(III-1), (III-2), (III-3), (III-4), (III-5), (III-6), (III-7), (III-8),(III-9), (III-10), (III-11), or (III-12), is a derivative of Amanitatoxin of Formula (I), the other one of either Drug₁ or Drug₂ can beselected from a protein, an antibody, antibody dimers, antibodymultimers, a bispecific or trispecific antibody, a single chainantibody, an antibody fragment that binds to the target cell, amonoclonal or polyclonal antibody, a chromophore molecule, a tubulysinderivative, a maytansinoid, a taxanoid (taxane), a CC-1065 analog, adaunorubicin or doxorubicin compound, a benzodiazepine dimer (e.g.,dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines), a calicheamicin or the enediyne antibiotic,an actinomycin, an azaserine, a bleomycin, a epirubicin, a tamoxifen, aidarubicin, a dolastatins, an auristatin derivative (e.g. monomethylauristatin E, MMAE, MMAF, auristatin PYE, auristatin TP, Auristatins2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), a duocarmycin, a thiotepa, avincristine, a hemiasterlin, a nazumamide, a microginin, a radiosumins,an alterobactin, a microsclerodermin, a theonellamide, an esperamicin, aPNU-159682, a geldanamycin, a methotrexate, a vindesines, a siRNA, anucleolytic enzyme. The examples of the preferred structures of some ofthese function molecules or cytotoxic drugs are illustrated below:

V-1, an antibody,

V-37, a siRNA, V-38, an enzyme or protein linked from N-terminal, V-39,an enzyme or protein linked from C-terminal.wherein R₁₀ are described in Formula (I),

is the site to link either linker L₁ or linker L₂.

In conjugation, the loading (drug/antibody ratio) and/or position of anADC with these derivatives of amanita toxins of this invention can becontrolled in other different ways, e.g., by: (i) limiting the molarexcess of drug-linker intermediate or linker reagent relative toantibody, (ii) limiting the conjugation reaction time or temperature,(iii) partial or limiting reductive conditions for cysteine thiolmodification, (iv) engineering by recombinant techniques the amino acidsequence of the antibody such that the number and position of lysine,carbohydrate, serine, tyrosine, glutamine, histidine, cysteine or theresidues is modified, or unnatural amino acids are inserted to theantibody sequences. These engineering methods are, but not limited,thioMab, thioFab, thiocarbohydrate, selenocysteine, cell-based approachof unnatural amino acids, open cell-free synthesis (OCFS) of unnaturalamino acids, oxidation or chemo-enzymatic modification of glycans,Formylglycine generating enzyme (FGE), Sortase or Transglutaminases,Conjugation via Fc-Binding Domains (FcBD) or via the Nucleotide BindingSite, or Catalytic antibodies) (Dennler, P. et al, Antibodies 2015, 4,197-224)

Other further examplary methods for preparing ADC of these derivativesof amanita toxins are described in FIGS. 1-28 and examples 1-70 in thedescription of the patent.

Treatments of Cell-Binding Agent-Drug Conjugates of these Derivatives ofAmanita Toxins

It is contemplated that the cell-binding agent—drug conjugate,preferably antibody-drug conjugates (ADC) of the present invention maybe used to treat various diseases or disorders, e.g. characterized bythe overexpression of a tumor antigen. Exemplary conditions orhyperproliferative disorders include benign or malignant tumors;leukemia and lymphoid malignancies. Others include neuronal, glial,astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal,blastocoelic, inflammatory, angiogenic and immunologic, includingautoimmune disorders.

In specific embodiment, the conjugates of the invention are used inaccordance with the compositions and methods of the invention for thetreatment of cancers. The cancers include, but are not limited,Adrenocortical Carcinoma, Anal Cancer, Bladder Cancer, Brain Tumor(Adult, Brain Stem Glioma, Childhood, Cerebellar Astrocytoma, CerebralAstrocytoma, Ependymoma, Medulloblastoma, Supratentorial PrimitiveNeuroectodermal and Pineal Tumors, Visual Pathway and HypothalamicGlioma), Breast Cancer, Carcinoid Tumor, Gastrointestinal, Carcinoma ofUnknown Primary, Cervical Cancer, Colon Cancer, Endometrial Cancer,Esophageal Cancer, Extrahepatic Bile Duct Cancer, Ewings Family ofTumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, IntraocularMelanoma, Gallbladder Cancer, Gastric Cancer (Stomach), Germ Cell Tumor,Extragonadal, Gestational Trophoblastic Tumor, Head and Neck Cancer,Hypopharyngeal Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cellcancer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, Acute Myeloid,Chronic Lymphocytic, Chronic Myelogenous, Hairy Cell), Lip and OralCavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell, Small Cell,Lymphoma (AIDS-Related, Central Nervous System, Cutaneous T-Cell,Hodgkin's Disease, Non-Hodgkin's Disease, Malignant Mesothelioma,Melanoma, Merkel Cell Carcinoma, Metasatic Squamous Neck Cancer withOccult Primary, Multiple Myeloma, and Other Plasma Cell Neoplasms,Mycosis Fungoides, Myelodysplastic Syndrome, MyeloproliferativeDisorders, Nasopharyngeal Cancer, Neuroblastoma, Oral Cancer,Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer (Epithelial, GermCell Tumor, Low Malignant Potential Tumor), Pancreatic Cancer (Exocrine,Islet Cell. Carcinoma), Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, Penile Cancer, Pheochromocytoma Cancer, PituitaryCancer, Plasma Cell Neoplasm, Prostate Cancer Rhabdomyosarcoma, RectalCancer, Renal Cell Cancer (kidney cancer), Renal Pelvis and Ureter(Transitional Cell), Salivary Gland Cancer, Sezary Syndrome, SkinCancer, Skin Cancer (Cutaneous T-Cell Lymphoma, Kaposi's Sarcoma,Melanoma), Small Intestine Cancer, Soft Tissue Sarcoma, Stomach Cancer,Testicular Cancer, Thymoma (Malignant), Thyroid Cancer, Urethral Cancer,Uterine Cancer (Sarcoma), Unusual Cancer of Childhood, Vaginal Cancer,Vulvar Cancer, Wilms' Tumor

In another specific embodiment, the compounds and the conjugates of theinvention are used in accordance with the compositions and methods ofthe invention for the treatment or prevention of an autoimmune disease.The autoimmune diseases include, but are not limited, AchlorhydraAutoimmune Active Chronic Hepatitis, Acute DisseminatedEncephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison'sDisease, Agammaglobulinemia, Alopecia areata, Amyotrophic LateralSclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis,Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopicallergy, Atopic Dermatitis, Autoimmune Aplastic Anemia, Autoimmunecardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis,Autoiminune inner ear disease, Autoimmune lymphoproliferative syndrome,Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmunepolyendocrine syndrome Types I, II, & III, Autoimmune progesteronedermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis,Balo di sease/Balo concentric sclerosis, Bechets Syndrome, Berger'sdisease, Bickerstaffs encephalitis, Blau syndrome, Bullous Pemphigoid,Castleman's disease, Chagas disease, Chronic Fatigue Immune DysfunctionSyndrome, Chronic inflammatory demyelinating polyneuropathy, Chronicrecurrent multifocal ostomyelitis, Chronic lyme disease, Chronicobstructive pulmonary disease, Churg-Strauss syndrome, CicatricialPemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease,Complement component 2 deficiency, Cranial arteritis, CREST syndrome,Crohns Disease (a type of idiopathic inflammatory bowel diseases),Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease,Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetesmellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler'ssyndrome, Discoid lupus erythematosus, Eczema, Endometriosis,Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysisbullosa acquisita, Erythema nodosum, Essential mixed cryoglobulinemia,Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia,Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinalpemphigoid, Giant cell arteritis, Glomerulonephritis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, Haemolytic anaemia,Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis suppurativa,Hughes syndrome (See Antiphospholipid syndrome), Hypogammaglobulinemia,Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonaryfibrosis, Idiopathic thrombocytopenic purpura (See Autoimmunethrombocytopenic purpura), IgA nephropathy (Also Berger's disease),Inclusion body myositis, Inflammatory demyelinating polyneuopathy,Interstitial cystitis, Irritable Bowel Syndrome, Juvenile idiopathicarthritis, Juvenile rheumatoid arthritis, Kawasaki's Disease,Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichenplanus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease(Also Amyotrophic lateral sclerosis), Lupoid hepatitis, Lupuserythematosus, Majeed syndrome, Meniere's disease, Microscopicpolyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease,Morphea, Mucha-Habermann disease, Muckle-Wells syndrome, MultipleMyeloma, Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy,Neuromyelitis optica (Devic's Disease), Neuromyotonia, Occularcicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord thyroiditis,Palindromic rheumatism, PANDAS (Pediatric Autoimmune NeuropsychiatricDisorders Associated with Streptococcus), Paraneoplastic cerebellardegeneration, Paroxysmal nocturnal hemoglobinuria, Parry Rombergsyndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus,Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis,POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progressive inflammatory neuropathy, Psoriasis, Psoriatic Arthritis,Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis,Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome,Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis,Rheumatoid fever, Sarcoidosis, Schizophrenia, Schmidt syndrome,Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syndrome,Spondyloarthropathy, Sticky blood syndrome, Still's Disease, Stiffperson syndrome, Subacute bacterial endocarditis, Susac's syndrome,Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu'sarteritis, Temporal arteritis (giant cell arteritis), Tolosa-Huntsyndrome, Transverse Myelitis, Ulcerative Colitis (a type of idiopathicinflammatory bowel diseases), Undifferentiated connective tissuedisease, Undifferentiated spondyloarthropathy, Vasculitis, Vitiligo,Wegener's granulotnatosis, Wilson's syndrome, Wiskott-Aldrich syndrome

In another specific embodiment, a binding molecule used for theconjugate for the treatment or prevention of an autoimmune diseaseincludes, but are not limited to, anti-elastin antibody; Abys againstepithelial cells antibody; Anti-Basement Membrane Collagen Type IVProtein antibody; Anti-Nuclear Antibody; Anti ds DNA; Anti ss DNA, AntiCardiolipin Antibody IgM, IgG; anti-celiac antibody; Anti PhospholipidAntibody IgK, IgG; Anti SM Antibody; Anti Mitochondrial Antibody;Thyroid Antibody; Microsomal Antibody, T-cells antibody; ThyroglobulinAntibody, Anti SCL-70; Anti-Jo; Anti-U.sub.1RNP; Anti-La/SSB; Anti SSA;Anti SSB; Anti Perital Cells Antibody; Anti Histones; Anti RNP; C-ANCA;P-ANCA; Anti centromere; Anti-Fibrillarin, and Anti GBM Antibody,Anti-ganglioside antibody; Anti-Desmogein 3 antibody; Anti-p62 antibody;Anti-sp100 antibody; Anti-Mitochondrial(M2) antibody; Rheumatoid factorantibody; Anti-MCV antibody; Anti-topoisomerase antibody;Anti-neutrophil cytoplasmic(cANCA) antibody.

In certain preferred embodiments, the binding molecule for the conjugatein the present invention, can bind to either a receptor or a receptorcomplex expressed on an activated lymphocyte which is associated with anautoimmune disease. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member (e.g. CD2, CD3, CD4, CD8, CD19,CD20, CD22, CD28, CD30, CD33, CD37, CD38, CD56, CD70, CD79, CD90,CD152/CTLA-4, PD-1, or ICOS), a TNF receptor superfamily member (e.g.CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-IBB, INF-R1, TNFR-2, RANK,TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3,TRAIL-R4, and APO-3), an integrin, a cytokine receptor, a chemokinereceptor, a major histocompatibility protein, a lectin (C-type, S-type,or I-type), or a complement control protein.

In another specific embodiment, useful binding ligands that areimmunospecific for a viral or a microbial antigen are humanized or humanmonoclonal antibodies. As used herein, the term “viral antigen”includes, but is not limited to, any viral peptide, polypeptide protein(e.g. HIV gp120, HIV nef, RSV F glycoprotein, influenza virusneuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplexvirus glycoprotein (e.g. gB, gC, gD, and gE) and hepatitis B surfaceantigen) that is capable of eliciting an immune response. As usedherein, the term “microbial antigen” includes, but is not limited to,any microbial peptide, polypeptide, protein, saccharide, polysaccharide,or lipid molecule (e.g. a bacteria, fungi, pathogenic protozoa, or yeastpolypeptide including, e.g., LPS and capsular polysaccharide 5/8) thatis capable of eliciting an immune response. Examples of antibodiesavailable 1 for the viral or microbial infection include, but are notlimited to, Palivizumab which is a humanized anti-respiratory syncytialvirus monoclonal antibody for the treatment of RSV infection; PRO542which is a CD4 fusion antibody for the treatment of HIV infection;Ostavir which is a human antibody for the treatment of hepatitis Bvirus; PROTVIR which is a humanized lgG.sub.1 antibody for the treatmentof cytomegalovirus; and anti-LPS antibodies.

The binding molecules-cytotoxic agent conjugates of this invention canbe used in the treatment of infectious diseases. These infectiousdiseases include, but are not limited to, Acinetobacter infections,Actinomycosis, African sleeping sickness (African trypanosomiasis), AIDS(Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax,Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever,Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Bacilluscereus infection, Bacterial pneumonia, Bacterial vaginosis, Bacteroidesinfection, Balantidiasis, Baylisascaris infection, BK virus infection,Black piedra, Blastocystis hominis infection, Blastomycosis, Bolivianhemorrhagic fever, Borrelia infection, Botulism (and Infant botulism),Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Buruliulcer, Calicivirus infection (Norovirus and Sapovirus),Campylobacteriosis, Candidiasis (Moniliasis; Thrush), Cat-scratchdisease, Cellulitis, Chagas Disease (American trypanosomiasis),Chancroid, Chickenpox, Chlamydia, Chlamydophila pneumoniae infection,Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficileinfection, Coccidioidomycosis, Colorado tick fever, Common cold (Acuteviral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease,Crimean-Congo hemorrhagic fever, Cryptococcosis, Cryptosporidiosis,Cutaneous larva migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirusinfection, Dengue fever, Dientamoebiasis, Diphtheria,Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever,Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection),Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythemainfectiosum (Fifth disease), Exanthem subitum, Fasciolopsiasis,Fasciolosis, Fatal familial insomnia, Filariasis, Food poisoning byClostridium perfringens, Free-living amebic infection, Fusobacteriuminfection, Gas gangrene (Clostridial myonecrosis), Geotrichosis,Gerstmann-Straussler-Scheinker syndrome, Giardiasis, Glanders,Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group Astreptococcal infection, Group B streptococcal infection, Haemophilusinfluenzae infection, Hand, foot and mouth disease (HFMD), HantavirusPulmonary Syndrome, Helicobacter pylori infection, Hemolytic-uremicsyndrome, Hemorrhagic fever with renal syndrome, Hepatitis A, HepatitisB, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex,Histoplasmosis, Hookworm infection, Human bocavirus infection, Humanewingii ehrlichiosis, Human granulocytic anaplasmosis, Humanmetapneumovirus infection, Human monocytic ehrlichiosis, Humanpapillomavirus infection, Human parainfluenza virus infection,Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono),Influenza, Isosporiasis, Kawasaki disease, Keratitis, Kingella kingaeinfection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease),Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis,Listeriosis, Lyme disease (Lyme borreliosis), Lymphatic filariasis(Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburghemorrhagic fever, Measles, Melioidosis (Whitmore's disease),Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis,Molluscum contagiosum, Mumps, Murine typhus (Endemic typhus), Mycoplasmapneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmianeonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD),Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis(South American blastomycosis), Paragonimiasis, Pasteurellosis,Pediculosis capitis (Head lice), Pediculosis corporis (Body lice),Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease,Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystispneumonia, Pneumonia, Poliomyelitis, Prevotella infection, Primaryamoebic meningoencephalitis, Progressive multifocal leukoencephalopathy,Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytialvirus infection, Rhinosporidiosis, Rhinovirus infection, Rickettsialinfection, Rickettsialpox, Rift Valley fever, Rocky mountain spottedfever, Rotavirus infection, Rubella, Salmonellosis, SARS (Severe AcuteRespiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis(Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola),Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection,Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringwormof the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium(Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis(Ocular Larva Migrans), Toxocariasis (Visceral Larva Migrans),Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworminfection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection,Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Viralpneumonia, West Nile Fever, White piedra (Tinea blanca), Yersiniapseudotuberculosis infection, Yersiniosis, Yellow fever, Zygomycosis.

The binding molecules, more preferred antibodies described in thispatent that are against pathogenic strains include, but are not limit,Acinetobacter baumannii, Actinomyces israelii, Actinomyces gerencseriaeand Propionibacterium propionicus, Trypanosoma brucei, HIV (Humanimmunodeficiency virus), Entamoeba histolytica, Anaplasma genus,Bacillus anthracis, Arcanobacterium haemolyticum, Junin virus, Ascarislumbricoides, Aspergillus genus, Astroviridae family, Babesia genus,Bacillus cereus, multiple bacteria, Bacteroides genus, Balantidium coli,Baylisascaris genus, BK virus, Piedraia hortae, Blastocystis hominis,Blastomyces dermatitides, Machupo virus, Borrelia genus, Clostridiumbotulinum, Sabia, Brucella genus, usually Burkholderia cepacia and otherBurkholderia species, Mycobacterium ulcerans, Caliciviridae family,Campylobacter genus, usually Candida albicans and other Candida species,Bartonella henselae, Group A Streptococcus and Staphylococcus,Trypanosoma cruzi, Haemophilus ducreyi, Varicella zoster virus (VZV),Chlamydia trachomatis, Chlamydophila pneumoniae, Vibrio cholerae,Fonsecaea pedrosoi, Clonorchis sinensis, Clostridium difficile,Coccidioides immitis and Coccidioides posadasii, Colorado tick fevervirus, rhinoviruses, coronaviruses, CJD prion, Crimean-Congo hemorrhagicfever virus, Cryptococcus neoformans, Cryptosporidium genus, Ancylostomabraziliense; multiple parasites, Cyclospora cayetanensis, Taenia solium,Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 andDEN-4)—Flaviviruses, Dientamoeba fragilis, Corynebacterium diphtheriae,Diphyllobothrium, Dracunculus medinensis, Ebolavirus, Echinococcusgenus, Ehrlichia genus, Enterobius vermicularis, Enterococcus genus,Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Humanherpesvirus 6 and Human herpesvirus 7, Fasciolopsis buski, Fasciolahepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily,Clostridium perfringens, Fusobacterium genus, Clostridium perfringens;other Clostridium species, Geotrichum candidum, GSS prion, Giardiaintestinalis, Burkholderia mallei, Gnathostoma spinigerum andGnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella granulomatis,Streptococcus pyogenes, Streptococcus agalactiae, Haemophilusinfluenzae, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71,Sin Nombre virus, Helicobacter pylori, Escherichia coli O157:H7,Bunyaviridae family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis CVirus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1,Herpes simplex virus 2, Histoplasma capsulatum, Ancylostoma duodenaleand Necator americanus, Hemophilus influenzae, Human bocavirus,Ehrlichia ewingii, Anaplasma phagocytophilum, Human metapneumovirus,Ehrlichia chaffeensis, Human papillomavirus, Human parainfluenzaviruses, Hymenolepis nana and Hymenolepis diminuta, Epstein-Barr Virus,Orthomyxoviridae family, Isospora belli, Kingella kingae, Klebsiellapneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Kuru prion,Lassa virus, Legionella pneumophila, Legionella pneumophila, Leishmaniagenus, Mycobacterium leprae and Mycobacterium lepromatosis, Leptospiragenus, Listeria monocytogenes, Borrelia burgdorferi and other Borreliaspecies, Wuchereria bancrofti and Brugia malayi, Lymphocyticchoriomeningitis virus (LCMV), Plasmodium genus, Marburg virus, Measlesvirus, Burkholderia pseudomallei, Neisseria meningitides, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Rickettsia typhi, Mycoplasma pneumoniae, numerous speciesof bacteria (Actinomycetoma) and fungi (Eumycetoma), parasitic dipterousfly larvae, Chlamydia trachomatis and Neisseria gonorrhoeae, vCJD prion,Nocardia asteroides and other Nocardia species, Onchocerca volvulus,Paracoccidioides brasiliensis, Paragonimus westermani and otherParagonimus species, Pasteurella genus, Pediculus humanus capitis,Pediculus humanus corporis, Phthinis pubis, Bordetella pertussis,Yersinia pestis, Streptococcus pneumoniae, Pneumocystis jirovecii,Poliovirus, Prevotella genus, Naegleria fowleri, JC virus, Chlamydophilapsittaci, Coxiella burnetii, Rabies virus, Streptobacillus monilifonnisand Spirillum minus, Respiratory syncytial virus, Rhinosporidiumseeberi, Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valleyfever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, Salmonellagenus, SARS coronavirus, Sarcoptes scabiei, Schistosoma genus, Shigellagenus, Varicella zoster virus, Variola major or Variola minor,Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus,Staphylococcus aureus, Streptococcus pyogenes, Strongyloidesstercoralis, Treponema pallidum, Taenia genus, Clostridium tetani,Trichophyton genus, Trichophyton tonsurans, Trichophyton genus,Epidermophyton floccosum, Trichophyton rubrum, and Trichophytonmentagrophytes, Trichophyton rubrum, Hortaea werneckii, Trichophytongenus, Malassezia genus, Toxocara canis or Toxocara cati, Toxoplasmagondii, Trichinella spiralis, Trichomonas vaginalis, Trichuristrichiura, Mycobacterium tuberculosis, Francisella tularensis,Ureaplasma urealyticum, Venezuelan equine encephalitis virus, Vibriocolerae, Guanarito virus, West Nile virus, Trichosporon beigelii,Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fevervirus, Mucorales order (Mucormycosis) and Entomophthorales order(Entomophthoramycosis), Pseudomonas aeruginosa, Campylobacter (Vibrio)fetus, Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis,Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonellatyphimurium, Treponema pertenue, Treponema carateneum, Borreliavincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae,Pneumocystis carinii, Brucella abortus, Brucella suis, Brucellamelitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Clamydia spp.; pathogenic fungi (Aspergillus furnigatus,Candida albicans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Trichomonas tenas, Trichomonas hominis, Tryoanosomagambiense, Trypanosoma rhodesiense, Leishmania donovani, Leishmaniatropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodiumvivax, Plasmodium falciparum, Plasmodium malaria); or Helminiths(Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium,and hookworms).

Other antibodies as a binding ligand in this invention for treatment ofviral disease include, but are not limited to, antibodies againstantigens of pathogenic viruses, including as examples and not bylimitation: Poxyiridae, Herpesviridae, Adenoviridae, Papovaviridae,Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae,influenza viruses, parainfluenza viruses, mumps, measles, respiratorysyncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae,Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae,Oncovirus [such as, HBV (Hepatocellular carcinoma), HPV (Cervicalcancer, Anal cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi'ssarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, Burkitt'slymphoma, Primary central nervous system lymphoma), MCPyV (Merkel cellcancer), SV40 (Simian virus 40), HCV (Hepatocellular carcinoma), HTLV-I(Adult T-cell leukemia/lymphoma)], Immune disorders caused virus: [suchas Human Immunodeficiency Virus (AIDS)]; Central nervous system virus:[such as, JCV (Progressive multifocal leukoencephalopathy), MeV(Subacute sclerosing panencephalitis), LCV (Lymphocyticchoriomeningitis), Arbovirus encephalitis, Orthomyxoviridae (probable)(Encephalitis lethargica), RV (Rabies), Chandipura virus, Herpesviralmeningitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomyelitis,Post-polio syndrome), HTLV-I (Tropical spastic paraparesis)];Cytomegalovirus (Cytomegalovirus retinitis, HSV (Herpetic keratitis));Cardiovascular virus [such as CBV (Pericarditis, Myocarditis)];Respiratory system/acute viral nasopharyngitis/viral pneumonia:[Epstein-Barr virus (EBV infection/Infectious mononucleosis),Cytomegalovirus; SARS coronavirus (Severe acute respiratory syndrome)Orthomyxoviridae: Influenzavirus A/B/C (Influenza/Avian influenza),Paramyxovirus: Human parainfluenza viruses (Parainfluenza), RSV (Humanrespiratory syncytial virus), hMPV]; Digestive system virus [MuV(Mumps), Cytomegalovirus (Cytomegalovirus esophagitis); Adenovirus(Adenovirus infection); Rotavirus, Norovirus, Astrovirus, Coronavirus;HBV (Hepatitis B virus), CBV, HAV (Hepatitis A virus), HCV (Hepatitis Cvirus), HDV (Hepatitis D virus), HEV (Hepatitis E virus), HGV (HepatitisG virus)]; Urogenital virus [such as, BK virus, MuV (Mumps)].

According to a further embodiment, the present invention also concernspharmaceutical compositions comprising the conjugate of the inventiontogether with a pharmaceutically acceptable carrier for treatment ofcancer and autoimmune disorders. The method for treatment of cancer,autoimmune disorders, infectious diseases or viral disease can bepracticed in vitro, in vivo, or ex vivo. Examples of in vitro usesinclude treatments of cell cultures in order to kill all cells exceptfor desired variants that do not express the target antigen; or to killvariants that express undesired antigen. Examples of ex vivo usesinclude treatments of hematopoietic stem cells (HSC) prior to theperformance of the transplantation (HSCT) into the same patient in orderto kill diseased or malignant cells. For instance, clinical ex vivotreatment to remove tumor cells or lymphoid cells from bone marrow priorto autologous transplantation in cancer treatment or in treatment ofautoimmune disease, or to remove T cells and other lymphoid cells fromallogeneic bone marrow or tissue prior to transplant in order to preventgraft-versus-host disease, can be carried out as follows. Bone marrow isharvested from the patient or other individual and then incubated inmedium containing serum to which is added the conjugate of theinvention, concentrations range from about 1 pM to 0.1 mM, for about 30minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation (=dose) are readily determined bythe skilled clinicians. After incubation the bone marrow cells arewashed with medium containing serum and returned to the patient by i.v.infusion according to known methods. In circumstances where the patientreceives other treatment such as a course of ablative chemotherapy ortotal-body irradiation between the time of harvest of the marrow andreinfusion of the treated cells, the treated marrow cells are storedfrozen in liquid nitrogen using standard medical equipment.

For clinical in vivo use, the cell binding agent-cytotoxic agentconjugates of this invention will be supplied as solutions or as alyophilized solid that can be redisolved in sterile water for injection.Examples of suitable protocols of conjugate administration are asfollows. Conjugates are given weekly, biweekly, triweekly or monthly for4-24 weeks or until disease progression or unacceptable toxicity as ani.v. bolus. Bolus doses are given in 10 to 500 ml of normal saline towhich human serum albumin (e.g. 0.5 to 1 mL of a concentrated solutionof human serum albumin, 100 mg/mL) can be added. Dosages will be about50 μs to 20 mg/kg of body weight weekly, biweekly, triweekly or monthlyi.v. (range of 10 μg to 200 mg/kg per injection). 4-24 weeks aftertreatment, the patient may receive a second course of treatment.Specific clinical protocols with regard to route of administration,excipients, diluents, dosages, times, etc., can be determined by theskilled clinicians. For example, the simple excipients can be0.002%-0.5% polysorbate (polysorbate 20, polysorbate 40, polysorbate 60,or polysorbate 80) or the other pharmaceutical acceptable sururfactant,such as sodium lauryl sulfate, triton X-100, 0.1%˜10% of binders, suchas saccharides and their derivatives (disaccharides: sucrose, lactose,trehalose or maltose), sugar alcohols such as xylitol, sorbitol ormaltitol, or polyethylene glycol, and 0.1%˜10% of pharmaceuticalbuffering agents such as citrate, succinate acetate, phosphate, orborate with a certain pH in the range of pH 4.5-9.5. In certainapplications, or a dose formula can contain other excipients, such aspolysaccharides and their derivatives: starches, cellulose or modifiedcellulose such as microcrystalline cellulose and cellulose ethers suchas hydroxypropyl cellulose (HPC), hypromellose (hydroxypropylmethylcellulose (HPMC)) or Hydroxypropyl cellulose; a protein such asgelatin, or albumin; Synthetic polymers: polyvinylpyrrolidone (PVP),polyethylene glycol (PEG); Antioxidants like vitamin A, vitamin E,vitamin C, retinyl palmitate, and selenium; The amino acids suchcysteine, tyrosine or methionine; Synthetic preservatives like theparabens: methyl paraben and propyl paraben.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any types of cancer, autoimmune diseases, graftrejections, and infections (viral, bacterial or parasite).

The amount of a conjugate which is required to achieve the desiredbiological effect, will vary depending upon a number of factors,including the chemical characteristics, the potency, and thebioavailability of the conjugates, the type of disease, the species towhich the patient belongs, the diseased state of the patient, the routeof administration, all factors which dictate the required dose amounts,delivery and regimen to be administered.

In general terms, the cell binding agent-cytotoxic agent conjugates ofthis invention may be provided in an aqueous physiological buffersolution containing 0.1 to 10% w/v conjugates for parenteraladministration. Typical dose ranges are from 1 mg/kg to 0.1 g/kg of bodyweight per day, per three-days, weekly, bi-weekly, tri-weekly or onceper four weeks. A preferred dose range is from 0.01 mg/kg to 20 mg/kg ofbody weight weekly, bi-weekly, tri-weekly or monthly, or an equivalentdose in a human child. The preferred dosage of drug to be administeredis likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, the formulation of the compound, the route of administration(intravenous, intramuscular, or other), the pharmacokinetic propertiesof the compound by the chosen delivery route, and the speed (bolus orcontinuous infusion) and schedule of administrations (number ofrepetitions in a given period of time).

The cell binding agent-cytotoxic agent conjugates of the presentinvention are also capable of being administered in unit dose forms,wherein the term “unit dose” means a single dose which is capable ofbeing administered to a patient, and which can be readily handled andpackaged, remaining as a physically and chemically stable unit dosecomprising either the active conjugate itself, or as a pharmaceuticallyacceptable composition, as described hereinafter. As such, typical totaldaily dose ranges are from 0.01 to 100 mg/kg of body weight. By way ofgeneral guidance, unit doses for humans range from 1 mg to 3000 mg perday. Preferably the unit dose range is from 0.1 to 500 mg administeredone to three times a day, weekly, bi-weekly, tri-weekly or monthly, andeven more preferably from 1 mg to 500 mg, weekly, bi-weekly, ortri-weekly, Conjugatess provided herein can be formulated intopharmaceutical compositions by admixture with one or morepharmaceutically acceptable excipients. Such unit dose compositions maybe prepared for use by oral administration, particularly in the form oftablets, simple capsules or soft gel capsules; or intranasally,particularly in the form of powders, nasal drops, or aerosols; ordermally, for example, topically in ointments, creams, lotions, gels orsprays, or via trans-dermal patches. The compositions may convenientlybe administered in unit dosage form and may be prepared by any of themethods well known in the pharmaceutical art, for example, as describedin Remington: The Science and Practice of Pharmacy, 21^(th) ed.;Lippincott Williams & Wilkins: Philadelphia, Pa., 2005. Preferredformulations include pharmaceutical compositions in which a compound ofthe present invention is formulated for oral or parenteraladministration. For oral administration, tablets, pills, powders,capsules, troches and the like can contain one or more of any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, or gum tragacanth; a diluent such asstarch or lactose; a disintegrant such as starch and cellulosederivatives; a lubricant such as magnesium stearate; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, or methylsalicylate. Capsules can be in the form of a hard capsule or softcapsule, which are generally made from gelatin blends optionally blendedwith plasticizers, as well as a starch capsule. In addition, dosage unitforms can contain various other materials that modify the physical formof the dosage unit, for example, coatings of sugar, shellac, or entericagents. Other oral dosage forms syrup or elixir may contain sweeteningagents, preservatives, dyes, colorings, and flavorings. In addition, theactive compounds may be incorporated into fast dissolve,modified-release or sustained-release preparations and formulations, andwherein such sustained-release formulations are preferably bi-modal.Preferred tablets contain lactose, cornstarch, magnesium silicate,croscarmellose sodium, povidone, magnesium stearate, or talc in anycombination. Liquid preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.The liquid compositions may also include binders, buffers,preservatives, chelating agents, sweetening, flavoring and coloringagents, and the like. Non-aqueous solvents include alcohols, propyleneglycol, polyethylene glycol, vegetable oils such as olive oil, andorganic esters such as ethyl oleate. Aqueous carriers include mixturesof alcohols and water, buffered media, and saline. In particular,biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be usefulexcipients to control the release of the active compounds. Intravenousvehicles can include fluid and nutrient replenishers, electrolytereplenishers, such as those based on Ringer's dextrose, and the like.Other potentially useful parenteral delivery systems for these activecompounds include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes.

Alternative modes of administration include formulations for inhalation,which include such means as dry powder, aerosol, or drops. They may beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Formulations for buccal administration include, forexample, lozenges or pastilles and may also include a flavored base,such as sucrose or acacia, and other excipients such as glycocholate.Formulations suitable for rectal administration are preferably presentedas unit-dose suppositories, with a solid based carrier, such as cocoabutter, and may include a salicylate. Formulations for topicalapplication to the skin preferably take the form of an ointment, cream,lotion, paste, gel, spray, aerosol, or oil. Carriers which can be usedinclude petroleum jelly, lanolin, polyethylene glycols, alcohols, ortheir combinations. Formulations suitable for transdermal administrationcan be presented as discrete patches and can be lipophilic emulsions orbuffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive.

In a specific embodiment, the cell binding agent-cytotoxic agentconjugates of this invention are administered concurrently with theother known or will be known therapeutic agents such as thechemotherapeutic agent, the radiation therapy, immunotherapy agents,autoimmune disorder agents, anti-infectious agents or the otherantibody-drug conjugates, resulting in a synergistic effect. In anotherspecific embodiment, the synergistic drugs or radiation therapy areadministered prior or subsequent to administration of a conjugate, inone aspect at least an hour, 12 hours, a day, a week, bi-week, tri-week,a month, in further aspects several months, prior or subsequent toadministration of a conjugate of the invention.

In other embodiments, the synergistic drugs include, but not limited to:

1). Chemotherapeutic agents: a). Alkylating agents: such as [Nitrogenmustards: (chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine,melphalan, trofosfamide); Nitrosoureas: (carmustine, lomustine); Alkylsulphonates: (busulfan, treosulfan); Triazenes: (dacarbazine); Platinumcontaining compounds: (carboplatin, cisplatin, oxaliplatin)]; b). PlantAlkaloids: such as [Vinca alkaloids: (vincristine, vinblastine,vindesine, vinorelbine); Taxoids: (paclitaxel, docetaxol)]; c). DNATopoisomerase Inhibitors: such as [Epipodophyllins:(9-aminocamptothecin, camptothecin, crisnatol, etoposide, etoposidephosphate, irinotecan, teniposide, topotecan,); Mitomycins: (mitomycinC)]; d). Anti-metabolites: such as {[Anti-folate: DHFR inhibitors:(methotrexate, trimetrexate); IMP dehydrogenase Inhibitors:(mycophenolic acid, tiazofurin, ribavirin, EICAR); Ribonucleotidereductase Inhibitors: (hydroxyurea, deferoxamine)]; [Pyrimidine analogs:Uracil analogs: (5-Fluorouracil, doxifluridine, floxuridine,ratitrexed(Tomudex)); Cytosine analogs: (cytarabine, cytosinearabinoside, fludarabine); Purine analogs: (azathioprine,mercaptopurine, thioguanine)]}; e). Hormonal therapies: such as{Receptor antagonists: [Anti-estrogen: (megestrol, raloxifene,tamoxifen); LHRH agonists: (goscrclin, leuprolide acetate);Anti-androgens: (bicalutamide, flutamide)]; Retinoids/Deltoids: [VitaminD3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol);Photodynamic therapies: (verteporfin, phthalocyanine, photosensitizerPc4, demethoxy-hypocrellin A); Cytokines: (Interferon-alpha,Interferon-gamma, tumor necrosis factor (TNFs), human proteinscontaining a TNF domain)]}; f). Kinase inhibitors, such as BIBW 2992(anti-EGFR/Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib,sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib.vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534),bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib,iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib,bevacizumab, cetuximab, Trastuzumab, Ranibizumab, Panitumumab,ispinesib; g). Others: such as gemcitabine, epoxomicins (e. g.carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide,tosedostat, zybrestat, PLX4032, STA-9090, Stimuvax, allovectin-7,Xegeva, Provenge, Yervoy, Isoprenylation inhibitors (such asLovastatin), Dopaminergic neurotoxins (such as1-methyl-4-phenylpyridinium ion), Cell cycle inhibitors (such asstaurosporine), Actinomycins (such as Actinomycin D, dactinomycin),Bleomycins (such as bleomycin A2, bleomycin B2, peplomycin),Anthracyclines (such as daunorubicin, doxorubicin (adriamycin),idarubicin, epirubicin, pirarubicin, zorubicin, mtoxantrone, MDRinhibitors (such as verapamil), Ca²⁺ ATPase inhibitors (such asthapsigargin), vismodegib, Histone deacetylase inhibitors (Vorinostat,Romidepsin, Panobinostat, Valproic acid, Mocetinostat (MGCD0103),Belinostat, PCI-24781, Entinostat, SB939, Resminostat, Givinostat,AR-42, CUDC-101, sulforaphane, Trichostatin A); Thapsigargin, Celecoxib,glitazones, epigallocatechin gallate, Disulfiram, Salinosporamide A.More detail lists of known and will be known anti-cancer drugs that canbe used as a combination therapy (a synergistic effect) with thecompounds and conjugates of the invention can be seen in National CancerInstitute (US) website (www.cancer.gov;www.cancer.gov/cancertopics/druginfo/alphalist), American Cancer Society(www.cancer.org/treatment/index) and Cancer Research UK(www.cancerrearchuk.org;(www.cancerresearchuk.org/cancer-help/about-cancer/treatment/cancer-drugs/)

2). An anti-autoimmune disease agent includes, but is not limited to,cyclosporine, cyclosporine A, aminocaproic acid, azathioprine,bromocriptine, chlorambucil, chloroquine, cyclophosphamide,corticosteroids (e.g. amcinonide, betamethasone, budesonide,hydrocortisone, flunisolide, fluticasone propionate, fluocortolonedanazol, dexamethasone, Triamcinolone acetonide, beclometasonedipropionate), DHEA, enanercept, hydroxychloroquine, infliximab,meloxicam, methotrexate, mofetil, mycophenylate, prednisone, sirolimus,tacrolimus.

3). An anti-infectious disease agent includes, but is not limited to,a). Aminoglycosides: amikacin, astromicin, gentamicin (netilmicin,sisomicin, isepamicin), hygromycin B, kanamycin (amikacin, arbekacin,bekanamycin, dibekacin, tobramycin), neomycin (framycetin, paromomycin,ribostamycin), netilmicin, spectinomycin, streptomycin, tobramycin,verdamicin; b). Amphenicols: azidamfenicol, chloramphenicol,florfenicol, thiamphenicol; c). Ansamycins: geldanamycin, herbimycin;d). Carbapenems: biapenem, doripenem, ertapenem, imipenem/cilastatin,meropenem, panipenem; e). Cephems: carbacephem (loracarbef),cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefaloridine,cefalotin or cefalothin, cefalexin, cefaloglycin, cefamandole,cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdaloxime, cefepime, cefminox, cefoxitin,cefprozil, cefroxadine, ceftezole, cefuroxime, cefixime, cefdinir,cefditoren, cefepime, cefetamet, cefmenoxime, cefodizime, cefonicid,cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cephalexin,cefpimizole, cefpiramide, cefpirome, cefpodoxime, cefprozil, cefquinome,cefsulodin, ceftazidime, cefteram, ceftibuten, ceftiolene, ceftizoxime,ceftobiprole, ceftriaxone, cefuroxime, cefuzonam, cephamycin (cefoxitin,cefotetan, cefmetazole), oxacephem (flomoxef, latamoxef); f).Glycopeptides: bleomycin, vancomycin (oritavancin, telavancin),teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. g.tigecycline; g). β-Lactamase inhibitors: penam (sulbactam, tazobactam),clavam (clavulanic acid); i). Lincosamides: clindamycin, lincomycin; j).Lipopeptides: daptomycin, A54145, calcium-dependent antibiotics (CDA);k). Macrolides: azithromycin, cethromycin, clarithromycin,dirithromycin, erythromycin, flurithromycin, josamycin, ketolide(telithromycin, cethromycin), midecamycin, miocamycin, oleandomycin,rifamycins (rifampicin, rifampin, rifabutin, rifapentine), rokitamycin,roxithromycin, spectinomycin, spiramycin, tacrolimus (FK506),troleandomycin, telithromycin; I). Monobactams: aztreonam, tigemonam;m). Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampicillin(pivampicillin, hetacillin, bacampicillin, metampicillin,talampicillin), azidocillin, azlocillin, benzylpenicillin, benzathinebenzylpenicillin, benzathine phenoxymethylpenicillin, clometocillin,procaine benzylpenicillin, carbenicillin (carindacillin), cloxacillin,dicloxacillin, epicillin, flucloxacillin, mecillinam (pivmecillinam),mezlocillin, meticillin, nafcillin, oxacillin, penamecillin, penicillin,pheneticillin, phenoxymethylpenicillin, piperacillin, propicillin,sulbenicillin, temocillin, ticarcillin; o). Polypeptides: bacitracin,colistin, polymyxin B; p). Quinolones: alatrofloxacin, balofloxacin,ciprofloxacin, clinafloxacin, danofloxacin, difloxacin, enoxacin,enrofloxacin, floxin, garenoxacin, gatifloxacin, gemifloxacin,grepafloxacin, kano trovafloxacin, levofloxacin, lomefloxacin,marbofloxacin, moxifloxacin, nadifloxacin, norfloxacin, orbifloxacin,ofloxacin, pefloxacin, trovafloxacin, grepafloxacin, sitafloxacin,sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin; q).Streptogramins: pristinamycin, quinupristin/dalfopristin); r).Sulfonamides: mafenide, prontosil, sulfacetamide, sulfamethizole,sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim,trimethoprim-sulfamethoxazole (co-trimoxazole); s). Steroidantibacterials: e.g. fusidic acid; t). Tetracyclines: doxycycline,chlortetracycline, clomocycline, demeclocycline, lymecycline,meclocycline, metacycline, minocycline, oxytetracycline,penimepicycline, rolitetracycline, tetracycline, glycylcyclines (e.g.tigecycline); u). Other types of antibiotics: annonacin, arsphenamine,bactoprenol inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine),dictyostatin, discodermolide, eleutherobin, epothilone, ethambutol,etoposide, faropenem, fusidic acid, furazolidone, isoniazid,laulimalide, metronidazole, mupirocin, mycolactone, NAM synthesisinhibitors (e. g. fosfomycin), nitrofurantoin, paclitaxel,platensirnycin, pyrazinamide, quinupristin/dalfopristin, rifampicin(rifampin), tazobactam tinidazole, uvaricin;

4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc, maraviroc,vicriviroc, gp41 (enfuvirtide), PRO 140, CD4 (ibalizumab); b). Integraseinhibitors: raltegravir, elvitegravir, globoidnan A; c). Maturationinhibitors: bevirimat, vivecon; d). Neuraminidase inhibitors:oseltamivir, zanamivir, peramivir; e). Nucleosides & nucleotides:abacavir, aciclovir, adefovir, amdoxovir, apricitabine, brivudine,cidofovir, clevudine, dexelvucitabine, didanosine (ddI), elvucitabine,emtricitabine (FTC), entecavir, famciclovir, fluorouracil (5-FU),3′-fluoro-substituted 2′, 3′-dideoxynucleoside analogues (e.g.3′-fluoro-2′,3′-dideoxythymidine (FLT) and3′-fluoro-2′,3′-dideoxyguanosine (FLG), fomivirsen, ganciclovir,idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. β-1-thymidine andβ-1-2′-deoxycytidine), penciclovir, racivir, ribavirin, stampidine,stavudine (d4T), taribavirin (viramidine), telbivudine, tenofovir,trifluridine valaciclovir, valganciclovir, zalcitabine (ddC), zidovudine(AZT); f). Non-nucleosides: amantadine, ateviridine, capravirine,diarylpyrimidines (etravirine, rilpivirine), delavirdine, docosanol,emivirine, efavirenz, foscarnet (phosphonoformic acid), imiquimod,interferon alfa, loviride, lodenosine, methisazone, nevirapine, NOV-205,peginterferon alfa, podophyllotoxin, rifampicin, rimantadine, resiquimod(R-848), tromantadine; g). Protease inhibitors: amprenavir, atazanavir,boceprevir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir,pleconaril, ritonavir, saquinavir, telaprevir (VX-950), tipranavir; h).Other types of anti-virus drugs: abzyme, arbidol, calanolide a,ceragenin, cyanovirin-n, diarylpyrimidines, epigallocatechin gallate(EGCG), foscarnet, griffithsin, taribavirin (viramidine), hydroxyurea,KP-1461, miltefosine, pleconaril, portmanteau inhibitors, ribavirin,seliciclib.

5). Other itnmunotheraphy drugs: e.g. imiquimod, interferons (e.g. α,β), granulocyte colony-stimulating factors, cytokines, Interleukins(IL-1˜IL-35), antibodies (e. g. trastuzumab, pertuzumab, bevacizumab,cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab,omalizumab), Protein-bound drugs (e.g., Abraxane), an antibodyconjugated with drugs selected from calicheamicin derivative, ofmaytansine derivatives (DM1 and DM4), CC-1065 and duocarmycin minorgroove binders, potent taxol derivatives, doxorubicin, auristatinantimitotic drugs (e. g. Trastuzumab-DM1, Inotuzumab ozogamicin,Brentuximab vedotin, Glembatumumab vedotin, lorvotuzumab mertansine,AN-152 LMB2, TP-38, VB4-845, Cantuzumab mertansine, AVE9633, SAR3419,CAT-8015 (anti-CD22), IMGN388, milatuzumab-doxorubicin, SGN-75(anti-CD70), Anti-CD22-MCC-DM1, IMGN853, Anti-CD22-MMAE,Anti-CD22-IVIMAF, Anti-CD22-calicheamicin.

The invention is further illustrated but not restricted by thedescription in the following examples.

EXAMPLES Example 1. Synthesis of(S)-3-(1H-indol-2-yl)-2-(tritylamino)propanoic Acid (1)

Chlorotrimethylsilane (3.4 mL, 26.9 mmol) was added slowly to asuspension of L-tryptophan (5.00 g, 24.5 mL) in methylene chloride (40mL) at r.t. The mixture was continuously stirred for 4.5 h andtriethylamine (6.8 mL, 49.0 mmol) was added, followed by a solution oftriphenylmethyl chloride (7.17 g, 25.7 mmol) in methylene chloride (20mL). The mixture was stirred at r.t. for 20 h and then quenched withmethanol (25 mL). The reaction was concentrated to near dryness andre-dissolved in methylene chloride, washed with 5% citric acid solution(3×) and brine. The organic phase was dried over anhydrous sodiumsulfate, filtered and concentrated. The residue was further dissolved inmethylene chloride and filtered over a celite pad and the filtrate wasconcentrated to give a pale white foam (11.8 g), which was used directlyin the next step. ESI MS m/z 446.5 ([M+H]⁺).

Example 2. Synthesis of (S)-methyl2-(3-(1H-indol-2-yl)-2-(tritylamino)propan amido)acetate (2)

To a solution of acid (9.27 g, 30.7 mmol) in THF (30 mL) was addedglycine methyl ester hydrochloride (2.85 g, 22.8 mmol) and HOBt (3.08 g,22.8 mmol). The mixture was cooled to 0° C. and triethylamine (7.4 mL,51.9 mmol) was added, followed by EDC.HCl (4.38 g, 22.8 mmol) inportions. The mixture was allowed to warm to r.t. and stirred for 20 hand then concentrated and redissolved in methylene chloride and washedwith 5% citric acid solution (3×) and brine. The organic phase was driedover anhydrous sodium sulfate, filtered and concentrated. The residuewas triturated with ethyl acetate and a white solid was collected byfiltration (6.46 g, 65% yield over two steps). ESI MS m/z 518.2([M+H]+).

Example 3. Synthesis of dimethyldioxirane (DMDO)

Distilled water (20 mL), acetone (30 mL), and NaHCO₃(24 g, 0.285 mol)were combined in a 1-L round-bottomed flask and chilled in an ice/waterbath with magnetic stirring for 20 min. After 20 min, stirring washalted and Oxone (25 g, 0.0406 mol) was added in a single portion. Theflask was loosely covered and the slurry was stirred vigorously for 15min while still submerged in the ice bath. The flask containing thereaction slurry was then attached to a rotary evaporator with a bath atroom temperature. The bump bulb (250 mL) was chilled in a dryice/acetone bath and a vacuum of 165 mtor was applied via a benchtopdiaphragm pump. After 15 min, the bath temperature was raised to 40° C.over 10 min. When the bath reached 40° C., the distillation was haltedimmediately via releasing the vacuum and raising the flask from theheated water bath. The pale yellow acetone solution of DMDO was decantedfrom the bump bulb directly into a graduated cylinder to measure thetotal volume of the solution (about 25 mL) and then the solution wasdried over Na2SO4.

The Na₂SO₄ is removed by filtration and rinsed with 5 mL of acetone.Titration of the obtained DMDO solution is then performed according tothe procedure of Adam, et al (Adam, W.; Chan, Y. Y.; Cremer, D.; Gauss,J.; Scheutzow, D.; Scheutzow, D.; Schindler, M. J. Org. Chem. 1987, 52,2800-2803). Results consistently showed 2.1-2.3 mmol of DMDO in thesolution. The DMDO solution was used immediately following titration.

Example 4. Synthesis of methyl2-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxamido)acetate (3)

To a solution of Trt-Trp-G1y-OMe (0.80 g, 1.54 mmol) in methylenechloride (20 mL) at −78° C. was added a solution of DMDO in acetone(2.25 mmol). After 1 h the mixture was concentrated to dryness underreduced pressure at room temperature. The crude material was purified bycolumn chromatography (hexanes/EtOAc/Et3N 70:30:1 to 30:70:1) to give alight yellow foam, the mixture of two diastereomers (0.58 g, 70% yield).ESI MS m/z 534.22 ([M+H]+).

Example 5. Synthesis of2-(3a-hydroxy-l-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxamido)acetic Acid (4)

To a solution of Tr-Hpi-Gly-OMe (mixture of diastereomers) (0.80 g, 1.50mmol) in dioxane/water (30 mL, v/v 2:1) was added LiOH (0.63 g, 15.0mmol) and the reaction was stirred at r.t. for 30 min (followingconsumption of the starting material by TLC (CH₂Cl₂/MeOH, 9:1)). Thereaction mixture was evaporated to dryness and the residue was purifiedby a short silica gel plug, eluting with CH₂Cl₂/MeOH/Et₃N (90:10:1).Fractions were combined to yield a light yellow solid as thetriethylamine salt of the two diastereomers (0.89 g, 95% yield).

Example 6. Synthesis of (5S,11R)-methyl5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-3,6,9-trioxo-11-((tritylthio)methyl)-2-oxa-4,7,10-triazadodecan-12-oate(5)

To a solution of Fmoc-Ile-Gly-OH (2.50 g, 6.09 mmol), H-Cys(Trt)-OMe(2.76 g, 7.30 mmol), HOBt (1.11 g, 7.30 mmol) in THF (40 mL) was addedDIPEA (2.6 mL, 15.3 mmol) at 0° C., followed by EDC.HCl (1.40 g, 7.30mmol) in portions. The reaction was warmed to r.t. and stirred for 16 h.The reaction was concentrated to dryness and diluted with ethyl acetateand washed with 5% citric acid (3×), saturated NaHCO₃(3×) and brine. Theorganic phase was dried over anhydrous Na₂SO₄ and concentrated. Theresidue was purified by column chromatography (0-20% ethylacetate/hexanes) to give a white solid (4.45 g, 95% yield). ESI MS m/z770.2 ([M+H]^(T)).

Example 7. Synthesis of (6S,12R)-methyl6-((S)-sec-butyl)-1-(3a-hydroxy-1- trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-2-yl)-1,4,7,10-tetraoxo-12-((tritylthio)methyl)-2,5,8,11-tetraazatridecan-13-oate(6)

Fmoc-Ile-Gly-Cys(Trt)-OMe (4.45 g, 5.78 mmol) was mixed withpiperidine/DMF (20%, 10 mL) and stirred at r.t. for 30 min. The reactionmixture was diluted with dichloromethane (50 mL) and washed with waterand brine. The organic phase was dried over anhydrous Na₂SO₄ andconcentrated. The residue was purified by column chromatography (0-10%MeOH/CH₂Cl₂ with 1% Et₃N) to give a white solid (3.12 g, 99% yield). ESIMS m/z 548.2 ([M+H]⁺).

The above solid (1.04 g, 1.90 mmol) was mixed with Trt-Hpi-Gly-OH.NEt₃(0.98 g, 1.58 mmol) in CH₂Cl₂ (10 mL), to which HOBt (0.29 g, 1.90 mmol)and DIPEA (0.7 mL, 4.00 mmol) were added at 0° C. EDC.HCl (0.36 g, 1.90mmol) was added at last in portions. The reaction was warmed to r.t. andstirred for 16 h and then diluted with ethyl acetate and washed with 5%citric acid (3×), saturated NaHCO₃ (3×) and brine. The organic phase wasdried over anhydrous Na₂SO₄ and concentrated. The residue was purifiedby column chromatography (20-70% ethyl acetate/hexanes) to give a whitesolid (0.75 g, 45% yield). ESI MS m/z 1049.4 ([M+H]⁺).

Example 8. Synthesis of(6S,12R)-6-((S)-sec-butyl)-1-(3a-hydroxy-1-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-2-yl)-1,4,7,10-tetraoxo-12-((tritylthio)methyl)-2,5,8,11-tetraazatridecan-13-oicAcid (7)

Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-OMe (0.75 g, 0.71 mmol) was dissolved indioxane/water (v/v 2:1, 30 mL) and treated with LiOH.H₂O (0.30 g, 7.1mmol) at r.t. for 30 min. The reaction mixture was evaporated to drynessand the residue was purified by a short silica gel plug, eluting withCH₂Cl₂/MeOH/Et₃N (90:10:1). Fractions were combined to yield a whitesolid as the triethylamine salt of Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-OH (0.77g, 95% yield).

Example 9. Synthesis of (2S,4R)-(9H-fluoren-9-yl)methyl4-(tert-butoxy)-2-(((2S,3S)-1-(tert-butoxy)-3-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carboxylate(8)

To a solution of Fmoc-Pro(O^(t)Bu)-OH (2.50 g, 6.10 mmol), H-Ile-O^(t)Bu(1.37 g, 7.32 mmol), HOBt (1.12 g, 7.32 mmol) in THF (40 mL) was addedDIPEA (2.6 mL, 15.3 mmol) at 0° C., followed by EDC.HCl (1.40 g, 7.32mmol) in portions. The reaction was warmed to r.t. and stirred for 16 h.After concentration, the residue was diluted with ethyl acetate andwashed with 5% citric acid (3×), saturated NaHCO₃ (3×) and brine. Theorganic phase was dried over anhydrous Na₂SO₄ and concentrated. Theresidue was purified by column chromatography (0-20% ethylacetate/hexanes) to give a white solid (2.55 g, 85% yield). ESI MS m/z579.3 ([M+H]⁺).

Example 10. Synthesis of (2S,3S)-tert-butyl2-(2S,4R)-1-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-methoxy-4-oxobutanoyl)-4-(tert-butoxy)pyrrolidine-2-carboxamido)-3-methylpentanoate(9)

Fmoc-Pro(O^(t)Bu)-Ile-O^(t)Bu (2.55 g, 4.40 mmol) was treated with 20%piperidine in DMF (20 mL) for 30 min. The reaction mixture wasconcentrated and purified by column chromatography (0-10% MeOH/CH₂Cl₂with 1% Et₃N) to give a white solid (1.41 g, 90% yield). ESI MS m/z357.2 ([M+H]⁺).

The above solid (1.41 g, 3.96 mmol) was mixed with Fmoc-Asp(OMe)-OH(1.22 g, 3.30 mmol) in DMF (20 mL), to which HOBt (0.61 g, 3.96 mmol)and DIPEA (1.4 mL, 8.25 mmol) were added at 0° C. EDC.HCl (0.76 g, 3.96mmol) was added at last in portions. The reaction was warmed to r.t. andstirred for 16 h and then diluted with ethyl acetate and washed withH₂O, 5% citric acid (3×), saturated NaHCO₃ (3×) and brine. The organicphase was dried over anhydrous Na₂SO₄ and concentrated. The residue waspurified by column chromatography (20-70% ethyl acetate/hexanes) to givea white solid (1.78 g, 76% yield). ESI MS m/z 708.4 ([M+H]⁺).

Example 11. Synthesis of ((6S,12R,15S)-methyl 15-((2S,4R)-4-(tert-butoxy)-2-(((2S,3S)-1-(tert-butoxy)-3-methyl-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-6-((S)-sec-butyl)-1-(3a-hydroxy-l-trityl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indol-2-yl)-1,4,7,10,13-pentaoxo-12-((tritylthio)methyl)-2,5,8,11,14-pentaazaheptadecan-17-oate(10)

Fmoc-Asp(OMe)-Pro(O^(t)Bu)-Ile-O^(t)Bu (0.59 g, 0.90 mmol) was mixedwith piperidine/DMF (20%, 10 mL) and stirred at r.t. for 30 min. Thereaction mixture was diluted with dichloromethane (50 mL) and washedwith water and brine. The organic phase was dried over anhydrous Na₂SO₄and concentrated. The residue was purified by column chromatography(0-10% MeOH/CH₂Cl₂ with 1% Et₃N) to give a white solid (0.43 g, 99%yield). ESI MS m/z 486.6 ([M+H]⁺).

The above solid (0.43 g, 0.89 mmol) was mixed withTrt-Hpi-Gly-Ile-Gly-Cys(Trt)-OH.NEt₃ (0.77 g, 0.67 mmol) in CH₂Cl₂ (5mL), to which HOBt (0.12 g, 0.80 mmol) and DIPEA (0.33 mL, 1.88 mmol)were added at 0° C. EDC.HCl (0.15 g, 0.80 mmol) was added at last inportions. The reaction was warmed to r.t. and stirred for 18 h and thendiluted with ethyl acetate and washed with 5% citric acid (3×),saturated NaHCO₃ (3×) and brine. The organic phase was dried overanhydrous Na₂SO₄ and concentrated. The residue was purified by columnchromatography (30-80% ethyl acetate/hexanes) to give a white solid(0.50 g, 50% yield). ESI MS m/z 1502.7 ([M+H]⁺).

Example 12. Synthesis of(2S,3S)-2-((2S,4R)-1-((S)-2-((3R,9S,15S)-15-amino-9-((S)-sec-butyl)-5,8,11,14-tetraoxo-2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,21-hexadecahydro-[1,4,7,10,13]thiatetraazacyclooctadecino[18,17-b]indole-3-carboxamido)-4-methoxy-4-oxobutanoyl)-4-hydroxypyrrolidine-2-carboxamido)-3-methylpentanoicacid (11)

Trt-Hpi-Gly-Ile-Gly-Cys(Trt)-Asp(OMe)-Pro(O^(t)Bu)-Ile-O^(t)Bu (0.50 g,0.33 mmol) was treated with neat TFA at r.t. for 5 h. Methanol was addedand the reaction was concentrated. This was repeated twice and theresidue was purified by prep-HPLC (H₂O/MeCN) to afford a white solid(99.6 mg, 34% yield).

Fmoc-Ile-OH was attached on the 2-chlorotrityl chloride resin accordingto the following protocol:

Fmoc-Ile-OH (0.35 g, 1.0 mmol) and DIPEA (0.70 mL, 4.0 mmol) weredissolved in dry methylene chloride (10 mL). The resulting solution wasadded to chlorotrityl resin (1.0 g, 0.911 mmol/g, GL Biochem) and themixture was shaken under nitrogen for 1.5 h. Subsequently methanol (2mL) was added and shaking continued for 30 min. The liquid was drainedunder vacuum and resin washed with methylene chloride (15 mL), DMF (10mL) and methanol (10 mL) and dried under vacuum.

Coupling was Performed According to the Following Protocol:

Resin was placed in a column and swollen in DMF (10 mL) for 30 min. Thesolvent was drained under vacuum and the N-terminal Fmoc protectinggroup was cleaved by shaking with 20% piperidine in DMF for 30 min.Following deprotection, the resin was washed with DMF (3×10 mL),followed by CH₂Cl₂ (3×10 mL) and again with DMF (3×10 mL). The next Fmocprotected amino acid (Fmoc-Xaa-OH, 5 eq.) was coupled to the resin withcoupling reagent HBTU (5 eq.) and DIPEA (10 eq.) in DMF (10 mL) withshaking for 2 h. The resin was then washed extensively with DMF (3×10mL), followed by CH₂Cl₂ (3×10 mL) and DMF (3×10 mL). A small sample wastaken and treated with hexafluoroisopropanol (HFIP) in CH₂Cl₂ for 5 minto cleave the peptide from the resin and checked by mass spectrometry.In case of coupling of non-commercially available amino acid, such asTrt-Hpi-Gly-OH, fewer equivalents (3 eq.) and longer time (3 h) wereemployed.

When all the couplings were completed, the resin-bounded peptide wastransferred to a round bottom flask and TFA (10 mL) was added andstirred at r.t. for 5 h. The acid labile protecting groups wereconcomitantly removed during TFA treatment. The resin was filtered andwashed with CH₂Cl₂ (10 mL) and methanol (10 mL). The filtrate wasconcentrated and partitioned between water and ethyl acetate. Theaqueous layer was purified by prep-HPLC (H₂O/MeCN) to yield a whitesolid of monocyclic octapeptide (40.3 mg, 5% yield). ESI MS m/z 888.38([M+H]⁺).

Example 13. Synthesis of Ile³-S-deoxo-amanitin (12)

To a solution of monocyclic octapeptide (25.7 mg, 0.0289 mmol) in dryDMF (5 mL) was added EDC.HCl (27.7 mg, 0.145 mmol), HOBt (39.0 mg, 0.289mmol) and DIPEA (0.025 mL, 0.145 mmol). The reaction was stirred at r.t.for 20 h and then concentrated and purified by prep-HPLC (H₂O/MeCN) togive a white solid compound 12 (9.0 mg, 36% yield). ESI MS m/z 870.40([M+H]⁺).

Example 14. Synthesis of Compound 13

To a solution of compound 12 (5.0 mg, 0.00575 mmol, 1.0 eq.) in THF (1mL) was added t-BuONO (7 μL, 0.0575 mmol) at 0° C. The reaction wasstirred at 0° C. for 1 h then room temperature 20 h. After water (5 mL)was added, the reaction mixture was concentrated and purified byprep-HPLC (H₂O/MeCN) to give a white solid (2.6 mg, 50% yield). ESI MSm/z 915.38 ([M+H]⁺).

Example 15. Synthesis of Compound 14

A mixture of nitro compound (2.6 mg, 0.00284 mmol) and Pd/C (10 wt %, 10mg) in methanol (2 mL) was hydrogenated (1 atm H₂) at r.t. for 1 h, andthen filtered through Celite (filter aid). The filtrate was concentratedto afford a white solid (2.5 mg, 99% yield). ESI MS m/z 885.38([M+1-1]⁺).

Example 16. Synthesis of Compound 15

To a solution of compound 14 (2.5 mg, 0.00283 mmol) and4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid (2.6 mg, 0.0141mmol) in dry DMF (1 mL) was added HATU (5.4 mg, 0.0141 mmol) and DIPEA(0.05 mL, 0.283 mmol). The reaction was stirred at r.t. for 20 h andthen diluted with ethyl acetate and washed with brine. The organic phasewas concentrated and purified by prep-HPLC (H₂O/MeCN) to give a whitesolid Ile³-S-deoxo-amanitin (1.7 mg, 56% yield). ESI MS m/z 1050.41([M+H]⁺).

Example 17. Synthesis of 2-(2-(dibenzylamino)ethoxy)ethanol (16)

2-(2-aminoethoxy)ethanol (21.00 g, 200 mmol, 1.0 eq.) and K₂CO₃(83.00 g,600 mmol, 3.0 eq.) in acetonitrile (350 mL) was added BnBr(57.0 mL, 480mmol, 2.4 eq.). The mixture was refluxed overnight. Water (1 L) wasadded and extracted with EtOAc (3×300 mL). The combined organic layerswere washed with brine (1000 mL), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (4:1hexanes/EtOAc) to give a colorless oil (50.97 g, 89.2% yield). MS ESIm/z [M+Na]⁺ 309.1967.

Example 18. Synthesis of tert-butyl3-(2-(2-(dibenzylamino)ethoxy)ethoxy) propanoate (17)

To a mixture of 2-(2-(dibenzylamino)ethoxy)ethanol (47.17 g, 165.3 mmol,1.0 eq.), tert-butyl acrylate (72.0 mL, 495.9 mmol, 3.0 eq.) and n-Bu₄NI(6.10 g, 16.53 mmol, 0.1 eq.) in DCM (560 mL) was added sodium hydroxidesolution (300 mL, 50%). The mixture was stirred overnight. The organiclayer was separated and the water layer was extracted with EtOAc (3×100mL). The organic layers were washed with water(3×300 mL) and brine (300mL), dried over anhydrous Na₂SO₄, filtered, concentrated and purified bySiO₂ column chromatography (7:1 hexanes/EtOAc) to give a colorless oil(61.08 g, 89.4% yield). MS ESI m/z [M+H]⁺ 414.2384.

Example 19. Synthesis of tert-butyl3-(2-(2-aminoethoxy)ethoxy)propanoate (18)

To a solution of tert-butyl 3-(2-(2-(dibenzylamino)ethoxy)ethoxy)propanoate (20.00 g, 48.36 mmol, 1.0 eq.) in THF (30 mL) and MeOH (60mL) was added Pd/C (2.00 g, 10 wt %, 50% wet) in a hydrogenation bottle.The mixture was shaken overnight, filtered through Celite (filter aid),and the filtrate was concentrated to afford a colorless oil (10.58 g,93.8% yield). MS ESI m/z [M+H]⁺ 234.1810.

Example 20. Synthesis of(E)-16-bromo-2,2-dimethyl-4,14-dioxo-3,7,10-trioxa-13-azaheptadec-15-en-17-oicAcid (19)

To a solution of 3-bromofuran-2,5-dione (89 mg, 0.5 mmol) in THF (5 mL),tert-butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (117 mg, 0.5 mmol) wasadded. The resulting solution was stirred at r.t. for 4 h. The solventwas removed under vacuum to afford compound 19 (205 mg, theoreticalyield). MS ESI m/z [M+H]⁻ 410.03.

Example 21. Synthesis of (E)-1-tert-butyl 18-methyl13-bromo-11,14-dioxo-4,7-dioxa-10,15-diazaoctadec-12-ene-1,18-dioate(20)

Compound 19 (205 mg, 0.5 mmol) and methyl 3-aminopropanoatehydrochloride (70 mg, 0.5 mmol) were dissolved in DCM (20 mL), to whichDIPEA (0.26 mL, 1.5 mmol) and EDC.HCl (144 mg, 0.75 mmol) were added.The resulting solution was stirred at r.t. overnight, and then washedwith brine (50 mL), dried over anhydrous Na₂SO₄. Concentration andpurification by column chromatography (0 to 10% MeOH/DCM) yieldedcompound 20 (88 mg, 36% yield). MS ESI m/z [M+H]⁺ 495.25.

Example 22. Synthesis of(E)-8-bromo-3,7,10-trioxo-2,14,17-trioxa-6,11-diazaicos-8-en-20-oic Acid(21)

Compound 20 (88 mg, 0.18 mmol) in DCM (3 mL) was treated with formicacid (6 ml) at 38° C. overnight. All volatiles were removed under vacuumto yield compound 21 (78 mg, theoretical yield).

Example 23. Synthesis of Compound 22

Compound 14 (2.0 mg, 0.00226 mmol) and compound 21 (5.0 mg, 0.0113 mmol)were dissolved in DMF (1 mL), to which HATU (4.3 mg, 0.0113 mmol) andDIPEA (2.0 μL, 0.0113 mmol) were added. The resulting solution wasstirred at r.t. overnight, diluted with ethyl acetate and then washedwith brine, dried over anhydrous Na₂SO₄. Concentration and purificationby prep-HPLC (H₂O/MeCN) gave a white solid (1.2 mg, 44% yield). MS ESIm/z [M+H]⁺1227.50.

Example 24. Synthesis of 4-(((benzyloxy)carbonyl)amino)butanoic Acid(23)

A solution of 4-aminobutyric acid (7.5 g, 75 mmol) and NaOH (6 g, 150mmol) in H₂O (40 mL) was cooled to 0° C. and treated with a solution ofCbzCl (16.1 g, 95 mmol) in THF (32 ml) dropwise. After 1 h, the reactionwas allowed to warm to r.t. and stirred for 3 h. THF was removed undervacuum, the pH of the aqueous solution was adjusted to 1.5 by additionof 6 N HCl. Extracted with ethyl acetate, and the organic layer waswashed with brine, dried and concentrated to give compound 23 (16.4 g,92% yield). MS ESI m/z [M+H]⁺ 238.08.

Example 25. Synthesis of tert-butyl4-(((benzyloxy)carbonyl)amino)butanoate (24)

DMAP (0.8 g, 6.56 mmol) and DCC (17.1 g, 83 mmol) were added to asolution of 4-(((benzyloxy)carbonyl)amino)butanoic acid (16.4 g, 69.2mmol) and t-BuOH (15.4 g, 208 mmol) in DCM (100 mL). After stirring atr.t. overnight, the reaction was filtered and filtrate concentrated. Theresidue was dissolved in ethyl acetate and the washed with 1N HCl, brineand dried over Na₂SO₄. Concentration and purification by columnchromatography (10 to 50% EtOAc/hexanes) yielded compound 24 (7.5 g, 37%yield). MS ESI m/z [M+Na]⁺ 316.13.

Example 26. Synthesis of tert-butyl 4-aminobutanoate (25)

tert-Butyl 4-(((benzyloxy)carbonyl)amino)butanoate (560 mg, 1.91 mmol)was dissolved in MeOH (50 mL), and mixed with Pd/C catalyst (10 wt %,100 mg) then hydrogenated (1 atm) at r.t. for 3 h. The catalyst wasfiltered off and all volatiles were removed under vacuum to affordcompound 25 (272 mg, 90% yield). MS ESI m/z [M+H]⁺ 160.13.

Example 27. Synthesis of(E)-2-bromo-4-((4-(tert-butoxy)-4-oxobutyl)amino)-4-oxobut-2-enoic Acid(26)

3-Bromofuran-2,5-dione (300 mg, 1.71 mmol) was dissolved in THF (20 mL),to which tert-butyl 4-aminobutanoate (272 mg, 1.71 mmol) was added andthe resulting solution was stirred at r.t. for 3 h. The solvent wasremoved under vacuum to afford compound 26 (572 mg, theoretical yield).MS ESI m/z [M+H]⁺ 338.04.

Example 28. Synthesis of (E)-tert-butyl 4-(3-bromo-4-((2-methoxyethyl)amino)-4-oxobut-2-enamido)butanoate (27)

2-Bromo-4((4-(tert-butoxy)-4-oxobutyl)amino)-4-oxobut-2-enoic acid(286mg, 0.85 mmol) and 2-methoxyethanamine (128 mg, 1.7 mmol) were dissolvedin DCM (40 mL), to which DIPEA (329 mg, 2.55 mmol) and EDC.HCl (490 mg,2.55 mmol) were added. The resulting solution was stirred at r.t. for 24h and then washed with brine, dried over Na₂SO₄. Concentration andpurification by column chromatography (0 to 10% MeOH/DCM) yieldedcompound 27 (102 mg, 31% yield). MS ESI m/z [M+H]⁺ 393.11.

Example 29. Synthesis of(E)-4-(3-bromo-4-((2-methoxyethyl)amino)-4-oxobut-2-enamido)butanoicacid (28)

Compound 27 (52 mg, 0.132 mmol) was dissolved in DCM (3 mL), to whichformic acid (6 ml) was added. The resulting solution was stirred at 38°C. overnight then concentrated to afford compound 28 (45 mg, theoreticalyield). MS ESI m/z [M+H]⁺ 339.05.

Example 30. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl4-(3-bromo-4-((2-methoxyethyl)amino)-4-oxobut-2-enamido)butanoate (29)

To a solution of compound 28 (45 mg, 0.132 mmol) in DCM (10 mL) NHS (23mg, 0.199 mmol) and EDC.HCl (38 mg, 0.199 mmol) were added. Afterstirring at r.t. for 3 h, the reaction was concentrated and purified bycolumn chromatography (10 to 50% EtOAc/hexanes) to yield compound 29 (57mg, 99% yield). MS ESI m/z [M+H]⁺ 436.06.

Example 31. Synthesis of Compound 30

To a solution of compound 14 (2.0 mg, 0.00226 mmol) in ethanol (1 mL)and phosphate buffer solution (pH 7.2, 1 mL) was added compound 29 (4.9mg, 0.0113 mmol) in ethanol (1 mL) over 30 min. After stirring at r.t.for 3 h, the reaction was concentrated and purified by prep-HPLC(H₂O/MeCN) to yield compound 30 (2.7 mg, 30% yield). MS ESI m/z [M+H]⁺1203.40.

Example 32. Synthesis of tert-butyl3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) propanoate (31)

To a solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanol (55.0 mL,410.75 mmol, 3.0 eq.) in anhydrous THF (200 mL) was added sodium (0.1g). The mixture was stirred until Na disappeared and then tert-butylacrylate (20.0 mL, 137.79 mmol, 1.0 eq.) was added dropwise. The mixturewas stirred overnight and then quenched by HCl solution (20.0 mL, 1 N)at 0° C. THF was removed by rotary evaporation, brine (300 mL) was addedand the resulting mixture was extracted with EtOAc (3×100 mL). Theorganic layers were washed with brine (3×300 mL), dried over anhydrousNa₂SO₄, filtered and concentrated to afford a colorless oil (30.20 g,79.0% yield), which was used without further purification. MS ESI m/z[M+H]⁺ 278.17.

Example 33. Synthesis of tert-butyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy) propanoate (32)

To a solution of tert-butyl 3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (30.20 g, 108.5 mmol, 1.0 eq.) and TsCl (41.37 g, 217.0 mmol,2.0 eq.) in anhydrous DCM (220 mL) at 0° C. was added TEA (30.0 mL,217.0 mmol, 2.0 eq.). The mixture was stirred at room temperatureovernight, and then washed with water (3×300 mL) and brine (300 mL),dried over anhydrous Na₂SO₄, filtered, concentrated and purified by SiO₂column chromatography (3:1 hexanes/EtOAc) to give a colorless oil (39.4g, 84.0% yield). MS ESI m/z [M+H]⁺ 433.28.

Example 34. Synthesis of tert-butyl3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy) propanoate (33)

To a solution of tert-butyl 3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (39.4 g, 91.1 mmol, 1.0 eq.) in anhydrous DMF(100 mL) wasadded NaN₃ (20.67 g, 316.6 mmol, 3.5 eq.). The mixture was stirred atroom temperature overnight. Water (500 mL) was added and extracted withEtOAc (3×300 mL). The combined organic layers were washed with water(3×900 mL) and brine (900 mL), dried over anhydrous Na₂SO₄, filtered,concentrated and purified by SiO₂ column chromatography (5:1hexanes/EtOAc) to give a light yellow oil (23.8 g, 85.5% yield). MS ESIm/z [M+Na] 326.20.

Example 35. Synthesis of tert-butyl3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) propanoate (34)

Raney-Ni (7.5 g, suspended in water) was washed with water (three times)and isopropyl alcohol (three times) and mixed with compound 33 (5.0 g,16.5 mmol) in isopropyl alcohol. The mixture was stirred under a H₂balloon at r.t. for 16 h and then filtered over a Celite pad, withwashing of the pad with isopropyl alcohol. The filtrate was concentratedand purified by column chromatography (5-25% MeOH/DCM) to give a lightyellow oil (2.60 g, 57% yield). MS ESI m/z [M+H]⁺ 279.19.

Example 36. Synthesis of di-tert-butyl14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioate(35)

Acetylenedicarboxylic acid (0.35 g, 3.09 mmol, 1.0 eq.) was dissolved inNMP (10 mL) and cooled to 0° C., to which compound 34 (2.06 g, 7.43mmol, 2.4 eq.) was added, followed by DMTMM (2.39 g, 8.65 mmol, 2.8 eq.)in portions. The reaction was stirred at 0° C. for 6 h and then dilutedwith ethyl acetate and washed with water and brine. The organic solutionwas concentrated and triturated with a mixture solvent of ethyl acetateand petroleum ether. The solid was filtered off and the filtrate wasconcentrated and purified by column chromatography (80-90% EA/PE) togive a light yellow oil (2.26 g, >100% yield), which was used withoutfurther purification. MS ESI m/z [M+H]⁺ 633.30.

Example 37. Synthesis of 14,17-dioxo-4,7,10,21,24,27-hexaoxa-13,18-diazatriacont-15-yne-1,30-dioic acid (36)

Compound 35 (2.26 g) was dissolved in dichloromethane (15 mL) and cooledto 0° C. then treated with TFA (15 mL). The reaction was warmed to r.t.and stirred for 45 min, and then the solvent and residual TFA wasremoved on rotovap. The crude product was purified by columnchromatography (0-15% MeOH/DCM) to give a light yellow oil (1.39 g, 86%yield for two steps). MS ESI m/z [M+H]+ 521.24.

Example 38. Synthesis of Compound 37

Compound 14 (2.0 mg, 0.00226 mmol) and compound 36 (5.9 mg, 0.0113 mmol)were dissolved in DMF (1 mL), to which HATU (4.3 mg, 0.0113 mmol) andDIPEA (2.0 μL, 0.0113 mmol) were added. The resulting solution wasstirred at r.t. overnight, and then concentrated and purified byprep-HPLC (H₂O/MeCN) to give a white solid (0.94 mg, 30% yield). MS ESIm/z [M+H]⁺ 1387.50.

Example 39. Synthesis of methyl2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (38)

To a solution of maleimide (6.35 g, 65.4 mmol) in ethyl acetate (120 mL)at 0° C. was added NMM (8.6 mL, 78.5 mmol), followed by methylchloroformate (6.0 mL, 78.5 mmol). The reaction was stirred at 0° C. for30 min and r.t. 1 h. The solid was filtered off and filtrate wasconcentrated. The residue was dissolved in methylene chloride andfiltered through a silica gel plug and eluted with methylene chloride toremove the red color. After concentration, the solid was triturated withethyl acetate and petroleum ether to give a white solid (9.0 g, 88%yield).

Example 40. Synthesis of tert-butyl(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl)carbamate (39)

A mixture of tert-butyl (2-aminoethyl)carbamate (11.2 mL) and saturatedNaHCO₃(120 mL) was stirred vigorously at 0° C., to which compound 38(10.0 g, 64.4 mmol) was added in portions. After stirring at 0° C. for30 min the reaction was warmed to r.t. and stirred for 1.0 h. The solidwas then collected by vacuum filtration and then dissolved in ethylacetate and washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated to give a white foam (13.6 g, 87% yield).

Example 41. Synthesis of tert-butyl(2-(1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)carbamate(40)

A mixture of compound 39 (6.0 g, 25.0 mmol) and furan (18 mL) in toluene(120 mL) was heated to 100° C. in a high pressure flask. The reactionwas stirred for 16 h and then the solvent removed. A solid was formedwhich was triturated with ethyl ether to give a white solid (6.5 g, 84%yield).

Example 42. Synthesis of2-(2-aminoethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dionehydrochloride (41)

To an ice cooled solution of compound 40 (7.90 g, 25.6 mmol) inmethylene chloride (60 mL) was added HCl/dioxane (10 mL of concentratedHCl dissolved in 30 mL of dioxane) slowly. The reaction mixture was thenwarmed to r.t. and stirred for 2 h. The process of reaction wasmonitored by TLC. Once completed, the reaction was concentrated andtriturated with ethyl acetate. A white solid was collected (6.32 g,theoretical yield) by vacuum filtration.

Example 43. Synthesis of Compound 42

Compound 14 (2.0 mg, 0.00226 mmol) and 3-((tert-butoxycarbonyl)amino)propanoic acid (2.1 mg, 0.0113 mmol) were dissolved in DMF (1 mL), towhich HATU (4.3 mg, 0.0113 mmol) and DIPEA (2.0 μL, 0.0113 mmol) wereadded. The resulting solution was stirred at r.t. overnight, and thenconcentrated and purified by prep-HPLC (H₂O/MeCN) to give a white solid(1.8 mg, 62% yield). MS ESI m/z [M+H]⁺ 1156.50.

Example 44. Synthesis of Compound 43

Compound 42 (1.8 mg, 0.0016 mmol) was dissolved in CH₂Cl₂ (2 mL) andcooled to 0° C., TFA (0.5 mL) was added dropwise. The reaction wasstirred at 0° C. for 5 h then diluted with CH₂Cl₂ and concentrated. Theresidue was purified by prep-HPLC (H₂O/MeCN) to give a white solid (1.1mg, 75% yield) as TFA sat. MS ESI m/z [M+H]⁺ 956.40.

Example 45. Synthesis of Compound 44

Compound 41 (0.40 g, 1.64 mmol) was suspended in methylene chloride (2.5mL) and cooled to 0° C. Phosphoryl trichloride (0.75 mL, 0.82 mmol) wasadded dropwise, followed by triethylamine (0.69 mL) slowly. The reactionwas stirred at 0° C. for 1 h and filtered over a celite pad. Thefiltrate was used immediately.

Compound 43 (1.1 mg, 0.00115 mmol) was dissolved in methylene chloride(0.5 mL) and cooled to 0 C, the above solution was added slowly. Afterstirring for 2 h, the reaction was concentrated and purified byprep-HPLC (H₂O/MeCN) to give a white solid (1.0 mg, 60% yield). MS ESIm/z [M+H]⁺ 1416.54.

Example 46. Synthesis of Compound 45

Compound 44 (1.0 mg, 0.000706 mmol) was dissolved in toluene (2 mL) andheated at 100° C. for 16 h. The solvent was then removed under vacuum togive a white solid (0.85 mg, 95% yield). MS ESI m/z [M+H]⁺ 1280.48.

Example 47. Synthesis of Compound di-tert-butyl1,2-bis(2-(tert-butoxy)-2-oxoethyl)hydrazine-1,2-dicarboxylate (46)

To a suspension of NaH (0.259 g, 6.48 mmol, 3.0 eq.) in anhydrous DMF (2mL) at room temperature was added di-tert-butylhydrazine-1,2-dicarboxylate (0.50 g, 2.16 mmol, 1.0 eq.) in anhydrousDMF (8 mL) in 10 minutes under nitrogen. The mixture was stirred at roomtemperature for 10 minutes and then cooled to 0° C. To which tert-butyl2-bromoacetate(1.4 mL, 8.61 mmol, 4.0 eq.) was added dropwise. Theresulting mixture was allowed to warm to room temperature and stirredovernight. Saturated ammonium chloride solution (100 mL) was added. Theorganic layer was separated and the aqueous layer was extracted withEtOAc (3×50 mL). The combined organic solution was washed with water andbrine, dried over anhydrous Na₂SO₄, concentrated and purified by SiO₂column chromatography (10:1 hexanes/EtOAc) to give compound 46 as acolorless oil (0.94 g, 99.6% yield). ESI MS m/z [M+Na]+483.4.

Example 48. Synthesis of Compound 2,2′-(hydrazine-1,2-diyl)diacetic Acid(47)

To a solution of compound 46 (0.94 g, 2.04 mmol) in DCM (4 mL) at 0° C.was added TFA (4 mL). The reaction was stirred for 30 minutes and thenwarmed to room temperature and stirred overnight. The mixture wasconcentrated, diluted with DCM, and concentrated. This operation wasrepeated for three times to give a white solid. Trituration with DCM anda white solid was collected by filtration (0.232 g, 76.8% yield). ESI MSm/z [M+H]⁺ 149.2.

Example 49. Synthesis of Compound2,2′-(1,2-bis(2-chloroacetyl)hydrazine-1,2-diyl)diacetic Acid (48)

To a solution of compound 47 (0.232 g, 1.57 mmol, 1.0 eq.) in anhydrousTHF (10 mL) at 0° C. was added 2-chloroacetyl chloride (0.38 mL, 4.70mmol, 3.0 eq.) in 10 minutes. The reaction was warmed to roomtemperature and stirred overnight and concentrated. The residue wasco-evaporated with THF for three times to give a white solid (0.472 g,theoretical yield). ESI MS m/z [M+H]⁺ 301.1.

Example 50. Synthesis of Compound 49

To a solution of compound 48 (0.0245 g, 0.0814 mmol) in anhydrous DCM (3mL) at 0° C. was added oxalyl dichloride (0.07 mL, 0.814 mmol) in 10minutes, followed by a drop of anhydrous DMF. The mixture was stirredfor 30 minutes before warmed to room temperature and stirred for 1.5 h.At last, the mixture was concentrated and co-evaporated with DCM forthree times to give a yellow oil, which was used directly.

To a solution of compound 43 (2.0 mg, 0.00209 mmol) in anhydrous DCM (1mL) was added one fourth of the above yellow oil in DCM (1 mL) at 0° C.The mixture was stirred for 2 h and then concentrated and purified byprep-HPLC (H₂O/MeCN) to give a colorless oil (1.3 g, 48% yield), whichwas used directly in the next step. ESI MS m/z [M+H]⁺ 1238.40.

Example 51. Synthesis of (E)-tert-butyl3-(2-(2-(3-bromoacrylamido)ethoxy) ethoxy)propanoate (50)

To a solution or (E)-3-bromoacrylic acid (0.15 g, 1 mmol), DMAP (0.15 g,1.2 mmol) and DCC (0.21 g, 1 mmol) in DCM (10 ml) at 0° C., tert-butyl3-(2-(2-aminoethoxy)ethoxy)propanoate (0.23 g, 1 mmol) were added. Thereaction mixture was allowed to warm to r.t. and stirred overnight. Thecrude product was concentrated and purified by SiO₂ columnchromatography with a gradient of EA/DCM to give the title product 50(0.31 g, 85% yield). ESI MS m/z [M+H]⁺ 366.08.

Example 52. Synthesis of (E)-3-(2-(2-(3-bromoacrylamido)ethoxy)ethoxy)propanoic Acid (51)

Compound 50 (0.31 g, 0.84 mmol) was dissolved in formic acid (4 ml) at0° C. then H₂O (2 ml) was added. The reaction mixture was allowed towarm to r.t. and stirred at r.t. overnight. The crude product wasconcentrated and used for the next step without further purification.ESI MS m/z [M+H]⁺ 310.03.

Example 53. Synthesis of (E)-2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-bromoacryl amido)ethoxy)ethoxy)propanoate (52)

Compound 51 (0.12 g, 0.39 mmol), NHS (0.067 g, 0.58 mmol) and EDC (0.11g, 0.58 mmol) were dissolved in DCM (10 ml), the mixture was stirred atr.t. overnight, concentrated and purified by SiO₂ column chromatographyto give the title product 52 (0.13 g, 82% yield). ESI MS m/z [M+H]⁺407.04.

Example 54. Synthesis of Compound 53

Compound 43 (2.0 mg, 0.00209 mmol), compound 52 (4.2 mg, 0.0105 mmol)were dissolved in DMA (1 ml), then phosphate buffer solution (pH 7.2, 1mL) was added. The mixture was stirred at r.t. overnight, concentratedand purified by reverse phase HPLC with a gradient of MeCN/H₂O to givethe title product 53 (0.9 mg, 33% yield). ESI MS m/z [M+H]⁺ 1248.40.

Example 55. Synthesis of Compound 54

To a solution of compound 14 (2.0 mg, 0.00226 mmol) in ethanol (1 mL)and phosphate buffer solution (pH 7.2, 1 mL) was added2,5-dioxopyrrolidin-1-yl2-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanamido)propanamido)acetate(4.6 mg, 0.0113 mmol) in ethanol (1 mL) over 30 min. After stirring atr.t. for 3 h, the reaction was concentrated and purified by prep-HPLC(H₂O/MeCN) to yield compound 54 (1.1 mg, 41% yield). MS ESI m/z [M+H]+1178.48.

Example 56. Synthesis of methyl4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (55)

Dimethyl succinate (20.0 g, 136.9 mmol) and dihydroxyethylamine (7.20 g,68.7 mmol) in the mixture of anhydrous toluene (500 ml) and pyridine (50ml) were refluxed at 150° C. for 28 h. The mixture was concentrated andpurified on SiO₂ column eluted with EtOAc/DCM (5%˜25% EtOAc) to affordthe title compound (12.5 g, 83% yield). ESI MS m/z [M+Na]⁺ 242.4.

Example 57. Synthesis of methyl 4-(bis(2-((methyl sulfonyl)oxy)ethyl)amino)-4-oxobutanoate (56)

Methyl 4-(bis(2-hydroxyethyl)amino)-4-oxobutanoate (12.0 g, 49.56 mmol)in anhydrous pyridine (350 ml) was added methanesulfonyl chloride (20.0g, 175.4 mmol). After stirred overnight the mixture was concentrated,diluted with EtOAc (350 ml), washed with cold 1 M NaH₂PO₄ (2×300 ml),dried over MgSO₄, filtered and evaporated to afford crude product (˜18.8g, >100% yield). The crude product was used for next step withoutfurther purification.

Example 58. Synthesis of 3,6-endoxo-Δ-tetrahydrophthalimide (57)

Maleimide (10.0 g, 103.0 mmol) in toluene (200 ml) was added furan (10.0ml, 137.4 mmol). The mixture was heated inside a 1 L of autoClave bombat 100° C. for 8 h. The bomb was cooled to room temperature, and theinside solid was rinsed with methanol, concentrated and crystallized inethyl acetate/hexane to afford 16.7 g (99%) of the title compound. 1HNMR (CDCl₃): 11.12 (s, 1H), 6.68-6.64 (m, 2H), 5.18-5.13 (m, 2H),2.97-2.92 (m, 2H). ESI MS m/z [M+Na]⁺ 188.04.

Example 59. Synthesis of Methyl4-((2-((3aR,4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)(2-((4R,7S,7aS)-1,3-dioxo-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl)amino)-4-oxobutanoate(58)

Methyl 4-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-oxobutanoate (56,fresh made, 90% pure, 8.5 g, ˜20 mmol) in DMA (350 ml) was added3,6-endoxo-A-tetrahydrophthalimide (10.2 g, 61.8 mmol), sodium carbonate(8.0 g, 75.5 mmol) and sodium iodide (0.3 g, 2.0 mmol). The mixture wasthen stirred at room temperature overnight, concentrated, diluted withEtOAc (350 ml), washed with sat'ed NaHCO₃ solution (300 ml), sat'ed NaClsolution (300 ml) and 1 M NaH₂PO₄ (300 ml). The organic layer was driedover Na₂SO₄, filtered, evaporated, loaded on SiO₂ column and eluted withEtOAc/hexane (10%˜30% EtOAc) to afford the title compound (7.9 g, 77%yield). ESI MS m/z [M+Na]⁺ 536.4.

Example 60. Synthesis of4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoicAcid (59)

Compound 58 (3.0 g, 5.8 mmol) and trimethylstannanol (4.8 g, 26.4 mmol)in 1,2-dichloroethane (150 ml) was refluxed at 80° C. for 8 h. It wascooled to room temperature and the residue was passed a short silica gelcolumn and eluted with dichloromethane/methanol to remove the extratrimethyltin hydroxide. Then the pooled fractions were combined,concentrated and diluted with DMA and toluene, refluxed at 120° C.overnight, loaded on SiO₂ column and eluted with MeOH/DCM (5%˜10% MeOH)to afford the title compound (1.62 g, 76% yield). ESI MS m/z [M+Na]⁺386.2.

Example 61. Synthesis of 2,5-dioxopyrrolidin-1-yl4-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutanoate(60)

To a solution or compound 59 (1.60 g, 4.4 mmol) in DMA (100 ml) wasadded NHS (0.76 g, 6.61 mmol) and EDC (1.70 g, 8.90 mmol). The mixturewas stirred overnight, evaporated and purified on SiO₂ column, elutedwith EtOAc/DCM (5% to 15% EtOAc) to afford the title product (1.72 g,85.0% yield). ESI MS m/z [M+Na]⁺483.2.

Example 62. Synthesis of Compound 61

To a solution of compound 14 (2.0 mg, 0.00226 mmol) in ethanol (1 mL)and phosphate buffer solution (pH 7.2, 1 mL) was added compound 60 (5.2mg, 0.0113 mmol) in ethanol (1 mL) over 30 min. After stirring at r.t.for 3 h, the reaction was concentrated and purified by prep-HPLC(H₂O/MeCN) to yield compound 61 (1.2 mg, 45% yield). MS ESI m/z[M+H]⁺1230.48.

Example 63. Synthesis of Compound 63

To a solution of compound 62 (5.0 mg, 0.00563 mmol, 1.0 eq.) in aceticacid (1 mL) and CH₂Cl₂ (1 mL) was added 70% HNO₃ (0.5 mL) at 0° C. Thereaction was stirred at 0° C. for 1 h then room temperature 2 h. Afterwater (5 mL) was added, the reaction mixture was concentrated andpurified by prep-HPLC (H₂O/MeCN) to give a light yellow solid (2.4 mg,46% yield). ESI MS m/z 931.34 ([M+H]⁺).

Example 64. Synthesis of Compound 64

Compound 63 (2.4 mg, 0.00257 mmol) in a mixture of CH₃CN (2 ml) and DMA(1 ml) was added DIPEA (9 μl, 0.0524 mmol) at 0° C. After stirred for 2min, POCl₃ (2.4 μl, 0.0262 mmol) was added dropwise at 0° C. The mixturewas stirred at r.t. for 2 h, and quenched with slowly addition of sat'edNaHCO₃ solution at 0° C. The mixture was concentrated and purifiedprep-HPLC (H₂O/MeCN) to give a white solid (2.0 mg, 77% yield). ESI MSm/z 1011.28 ([M+H]⁺).

Example 65. Synthesis of Compound 65

A mixture of compound 64 (2.0 mg, 0.00197 mmol) and Pd/C (10 wt %, 5 mg)in methanol (2 mL) was hydrogenated (1 atm H₂) at r.t. for 1 h, and thenfiltered through Celite (filter aid). The filtrate was concentrated toafford a white solid (1.8 mg, 93% yield). ESI MS m/z 981.33 ([M+H]⁺).

Example 66. Synthesis of Compound 66

To a solution of compound 14 (1.8 mg, 0.00183 mmol) in ethanol (1 mL)and phosphate buffer solution (pH 7.2, 1 mL) was added2,5-dioxopyrrolidin-1-yl4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate (2.6 mg, 0.0930 mmol)in ethanol (1 mL) over 10 min. After stirring at r.t. for 3 h, thereaction was concentrated and purified by prep-HPLC (H₂O/MeCN) to yieldcompound 30 (1.3 mg, 62% yield). MS ESI m/z [M+H]⁺ 1146.45.

Example 67. General Method of Conjugation of Compound 15, 22, 30, 37,45, 49, 53, 54, 61, or 66 Independently to a Her2 Antibody (Herceptin)

To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0˜8.0, were addedof 0.70˜2.0 mL PBS buffer of 100 mM NaH₂PO₄, pH 6.5˜8.5 buffers, TCEP(14-35 μL, 20 mM in water) and the compound 15, 22, 30, 37, 45, 49, 53,54, 61, or 66 (14-28 μL, 20 mM in DMA). The mixture was incubated at RTfor 4˜16 h, then DHAA (135 μL, 50 mM) was added in. After continuousincubation at RT overnight, the mixture was purified on G-25 columneluted with 100 mM NaH₂PO₄, 50 mM NaCl pH 6.0˜7.5 buffer to afford12.0˜18.4 mg of the conjugate compound A1, A2, A3, A4, A5, A6, A7, A8,A9, or A10 (68%˜83% yield) accordingly in 13.0˜15.8 ml buffer. Thedrug/antibody ratio (DAR) was 1.9˜4.0, which was determined viaUPLC-QTOF mass spectrum. It was 94˜99% monomer analyzed by SEC HPLC(Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID×30 cm, 0.5 ml/min, 100 min)and a single band measured by SDS-PAGE gel.

Example 68. General Method of Conjugation of Compound 22, 30, 37, 45,49, 54 or 61 Independently to Herceptin (a Her2 Antibody)

To a mixture of 2.0 mL of 10 mg/ml Herceptin in pH 6.0-8.0, 0.70-2.0 mLof 100 mM NaH₂PO₄, 1 mM Na₂SO₃, pH 6.5˜8.5 buffer, and the compound 22,30, 37, 45, 49, 54 or 61 independently (21-24 μL, 20 mM in DMA)incubated for 1 h, was added TCEP (14-23 μL, 20 mM in water). After themixture was continued to incubate at RT for 8˜24 h, DHAA (135 μL, 50 mM)was added in. And the solution was continuously incubated for another 12h. then purified on G-25 column eluted with 100 mM NaH₂PO₄, 50 mM NaClpH 6.0˜7.5 buffer to afford 13.8˜17.6 mg of the conjugate compound A2,A3, A4, A5, A6, A7 or A9 respectively (63%˜87% yield) in 13.6˜15.7 mlbuffer. The drug/antibody ratio (DAR) was 1.9˜3.8, which was determinedvia UPLC-QTOF mass spectrum. It was 96˜99% monomer analyzed by SEC HPLC(Tosoh Bioscience, Tskgel G3000SW, 7.8 mm ID×30 cm, 0.5 ml/min, 100 min)and a single band measured by SDS-PAGE gel or two bands when a reducereagent DTT was in the SDS Page.

Example 69. In Vitro Cytotoxicity Evaluation of Conjugates A1, A2, A3,A4, A5, A6, A7, A8, A9, and A10 in Comparison with T-DM1

The cell line used in the cytotoxicity assays was NCI-N87, a humangastric carcinoma cell line; The cells were grown in RPMI-1640 with 10%FBS. To run the assay, the cells (180 μl, 6000 cells) were added to eachwell in a 96-well plate and incubated for 24 hours at 37° C. with 5%CO₂. Next, the cells were treated with test compounds (20 μl) at variousconcentrations in appropriate cell culture medium (total volume, 0.2mL). The control wells contain cells and the medium but lack the testcompounds. The plates were incubated for 120 hours at 37° C. with 5%CO₂. MTT (5 mg/ml) was then added to the wells (200) and the plates wereincubated for 1.5 hr at 37° C. The medium was carefully removed and DMSO(180 μl) was added afterward. After it was shaken for 15 min, theabsorbance was measured at 490 nm and 570 nm with a reference filter of620 nm. The inhibition % was calculated according to the followingequation: inhibition %=[1-(assay-blank)/(control-blank)]×100.

The cytotoxicity results:

DAR (drug ratio) N87 cell (Ag+) IC₅₀ (nM) Conjugate A1 3.8  1.19 nMConjugate A2 2.9  3.32 nM Conjugate A3 2.8 17.33 nM Conjugate A4 2.769.24 nM Conjugate A5 3.2  3.13 nM Conjugate A6 2.7  53.3 nM ConjugateA7 2.9  5.53 nM Conjugate A8 3.2 48.13 nM Conjugate A9 2.3  5.71 nMConjugate A10 2.8  3.63 nM T-DM1 3.5  0.19 nM

The conjugates with the bridge linkers were less potent than T-DM1 invitro.

Example 70. Antitumor Activity In Vivo

The in vivo efficacy of conjugates A1, A2, A3, A5, A6, A7, A9, and A10along with T-DM1 were evaluated in a human gastric carcinoma N-87 cellline tumorxenograft models. Five-week-old female BALB/c Nude mice (60animals) were inoculated subcutaneously in the area under the rightshoulder with N-87 carcinoma cells (5×10⁶ cells/mouse) in 0.1 mL ofserum-free medium. The tumors were grown for 8 days to an average sizeof 123 mm³. The animals were then randomly divided into 10 groups (6animals per group). The first group of mice served as the control groupand was treated with the phosphate-buffered saline vehicle. Theremaining 9 groups were treated with conjugates A1, A2, A3, A5, A6, A7,A9, and A10 and T-DM1 respectively at dose of 6 mg/Kg administeredintravenously. Three dimensions of the tumor were measured every 4 daysand the tumor volumes were calculated using the formula tumor volume=1/2(length×width×height). The weight of the animals was also measured atthe same time. A mouse was sacrificed when any one of the followingcriteria was met: (1) loss of body weight of more than 20% frompretreatment weight, (2) tumor volume larger than 1500 mm³, (3) too sickto reach food and water, or (4) skin necrosis. A mouse was considered tobe tumor-free if no tumor was palpable.

The results were plotted in FIG. 29. All the 9 conjugates did not causethe animal body weight loss. And the animals at control group weresacrificed at day 30 due to the tumor volume larger than 1200 mm³ andthey were too sick. In FIG. 30. All 6/6 animals at the groups ofcompounds A2, A5, A7, A9 and A10 had completely no tumor measurable atday 16 till day 62 (the end of experiment). In contrast T-DM1 at dose of6 mg/Kg was not able to eliminate the tumors completely and it onlyinhibited the tumor growth for 37 days. Conjugate compounds A1, A3, andA6 also did not eradicate the tumor completely at dose of 6 mg/Kg, buthad better antitumor activity than T-DM1. More importantly, all animalstreated with conjugate compounds A1, A2, A3, A5, A6, A7, A9, and A10 hadno or less liver toxicities than the animals treated with T-DM1 whenmeasured levels of alanine aminotransferase (ALT) and aspartameaminotransferase (AST) in serum at the end of experiment (data notshown). This demonstrates that the conjugates of this patent applicationwould have broader therapeutical applications than the traditionalconjugates.

The invention claimed is:
 1. A compound of Formula (I)

or a pharmaceutically acceptable salt, hydrate, or hydrated saltthereof; or a polymorphic crystalline structures thereof; or an opticalisomer, racemate, diastereomer or enantiomer thereof, wherein -----represents a single bond that links any carbon position of the aromatic(indole) ring;

represents an optional single bond or an absent bond; R₁ and R₂ areindependently selected from the group consisting of H, OH, CH₂OH,CH(OH)CH₂OH, CH(CH₃)CH₂OH, CH(OH)CH₃, C₁-C₈ alkyl, —OR₁₂ (ether), C₂-C₈alkenyl, alkynyl, heteroalkyl, —OCOR₁₂ (ester), —OC(═O)OR₁₂ (carbonate),—OC(═O)NHR₁₂ (carbamate); C₃-C₈ aryl, heterocyclic, carbocyclic,cycloalkyl, heterocycloalkyl, heteroaralkyl, and alkylcarbonyl; R₃ andR₄ are independently selected from the group consisting of H, OH, —OR₁₂(ether), —OCOR₁₂ (ester), —OCOCH₃ (acetate), —OCOOR₁₂ (carbonate),—OC(═O)NHR₁₂ (carbamate), —OP(O)(OR₁₂)(OR₁₂′) (phosphate),OP(O)(NHR₁₂)(NHR₁₂′) (phosphamide), O—SO₃ ⁻, and O-glycoside; R₅ isselected from the group consisting of H, OH, NH₂, NHOH, NHNH₂, —OR₁₂,—NHR₁₂, NHNHR₁₂, —NR₁₂R₁₂′, and N(H)(R₁₂)R₁₃CO(Aa)_(p), wherein Aa is anamino acid or a polypeptide, p represents 0-6; R₆ is selected from thegroup consisting of H, OH, CH₂OH, CH(OH)CH₂OH, CH(CH₂OH)₂, CH(CH₃)OH,CH₂CH₂OH, PrOH, BuOH, C₁˜C₈ alkyl, —OR₁₂ (ether), C₂˜C₈ alkenyl,alkynyl, heteroalkyl, or —OCOR₁₂ (ester); and C₃˜C₈ aryl, heterocyclic,or carbocyclic; R₇, R₈ and R₉ are independently selected from the groupconsisting of H, OH, CH₃, CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂OH, CH(OH)CH₂OH,CH₂CH(OH)CH₂OH, CH(CH₂OH)₂, CH₂C(OH)(CH₂OH)₂, CH₂C(OH)(CH₃)(CH₂OH),CH₂C(OH)(CH(CH₃)₂)(CH₂OH), CH₂CH₂OH, PrOH, BuOH, CH₂COOH, CH₂CH₂COOH,CH(OH)COOH, CH₂CONH₂, CH₂CH₂CONH₂, CH₂CH₂CH₂CH₂NH₂,CH₂CH₂CH₂NHC(═NH)NH₂, C₁˜C₈ alkyl, CH₂Ar, CH₂SH, CH₂SR₁₂, CH₂SSR₁₂,CH₂SSAr, CH₂CH₂SCH₃, —OR₁₂ (ether), C₂˜C₈ alkenyl, alkynyl, heteroalkyl,or —OCOR₁₂ (ester); and C₃˜C₈ aryl, heterocyclic, or carbocyclic; R₁₀ isselected from the group consisting of H, NH₂, OH, SH, NO₂, halogen,—NHOH, —N₃ (azido); —CN (cyano); C₁˜C₈ alkyl, C₂˜C₈ alkenyl, alkynyl,heteroalkyl; C₃˜C₈ aryl, heterocyclic, or carbocyclic; —OR₁₂ (ether),—OCOR₁₂ (ester), —OCOCH₃ (acetate), —OC(O)OR₁₂ (carbonate),—OC(O)CH(R₁₂)NHAa (Aa is an amino acid group), —NR₁₂R₁₂′ (amine),—NR₁₂COR₁₂′ (amine), —NR₁₂NR₁₂′NR₁₂″ (amine); —OCONR₁₂R₁₂′(carbamate);—NR₁₂(C═NH)NR₁₂′R₁₂″ (guanidinum); —NR₁₂CO(Aa)_(p), (an amino acid orpeptide, wherein Aa is an amino acid or a polypeptide, p represents0-6); —N(R₁₂)CONR₁₂′R₁₂″ (urea); —OCSNHR₁₂ (thiocarbamate); —SH (thiol);—SR₁₂ (sulfide); —S(O)R₁₂ (sulfoxide); —S(O₂)R₁₂ (sulfone); —SO₃, HSO₃,HSO₂, or a salt of HSO₃ ⁻, SO₃ ²⁻ or —HSO₂ ⁻(sulphite), —OSO₃ ⁻;—N(R₁₂)SOOR₁₂′ (sulfonamide); H₂S₂O₅ or a salt of S₂O₅ ²⁻(metabisulfite); PO₃SH₃, PO₂S₂H₂, POS₃H₂, PS₄H₂ or a salt of PO₃S³⁻,PO₂S₂ ³⁻, POS₃ ³⁻, PS₄ ³⁻ (mono-, di-, tri-, and tetra-thiophosphate);(R₁₂O)₂POSR₁₂′ (thiophosphate ester); HS₂O₃ or a salt of S₂O₃ ²⁻(thiosulfate); HS₂O₄ or a salt of S₂O₄ ²⁻ (dithionite);(P(═S)(OR₁₂)(S)(OH) or a salt formed with a cation (phosphorodithioate);—N(R₁₂)OR₁₂′ (hydroxylamine derivative); R₁₂C(═O)NOH or a salt formedwith a cation (hydroxamic acid); (HOCH₂SO₂ ⁻, or its salt (formaldehydesulfoxylate); —N(R₁₂)COR₁₂′ (amide); R₁₂R₁₂′R₁₂″NPO₃H(trialkylphosphor-amidate or phosphoramidic acid); ArAr′Ar″NPO₃H(triarylphosphonium); OP(O)(OM₁)(OM₂), OCH₂OP(O)(OM₁)(OM₂), OSO₃M₁;O-glycoside (glucoside, galactoside, mannoside, glucuronoside, alloside,fructoside), NH-glycoside, S-glycoside and CH₂-glycoside; M₁ and M₂ areindependently H, Na, K, Ca, Mg, NH₄, or NR₁′R₂′R₃′; R₁′, R₂′ and R₃′ areindependently H, or alkyl; and Ar, Ar′, and Ar″ are C₃-C₈ aryl orheteroaromatic group; R₁₁ is H, C₁˜C₈ alkyl; C₂˜C₈ alkenyl, alkynyl, orheteroalkyl; or C₃˜C₈ aryl or heteroaryl; R₁₂, R₁₂′, and R₁₂″ areindependently selected from the group consisting of H, C₁˜C₈ alkyl;C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl, heteroaryl,heterocyclic, and carbocyclic; X is S, O, NH, SO, SO₂, or CH₂; m is 0 or1; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20; L is a releasable linker having the formula of:-Ww-(Aa)r-Tt-; or -Ww-(Aa)r-Tt-Q; or Q-Ww-(Aa)r-Tt-; wherein W is aStretcher unit; w is 0 or 1; Aa is an amino acid unit comprisingindependent amino acids; r is an integer ranging from 0 to 100, theStretcher unit W comprises a self-immolative or a non-self-immolativecomponent, peptidyl unit, a hydrazone bond, a disulfide, an ester, anoxime, an amide, or a thioether bond, the self-immolative componentcomprises a 2-aminoimidazol-5-methanol compound, heterocyclic PABanalog, beta-glucuronide, and ortho or para-aminobenzylacetal; thenon-self-immolative component is one of the following structures:

wherein the (*) atom is a point of attachment of additional spacer orreleasable linker, a cytotoxic agent, or a cell-binding agent; X¹, Y¹,Q¹, R₁₂, R₁₂′ are defined as above; r is 0˜100; p and q is independently0, 1, 2, 3, 4, 5 or 6; Spacer (T) is a linear, branched or cyclic alkyl,alkenyl, alkynyl or aryl having from 1 to 10 carbon atoms, or apolyethylene glycol (—CH₂CH₂O—) spacer; t is 0, or 1˜100, or T undergoescyclization upon amide bond hydrolysis, of a substituted andunsubstituted 4-aminobutyric acid amide, substituted bicycle [2.2.1] andbicycle [2.2.2] ring systems or 2-aminophenylpropionic acid amide; Q isa cell-binding agent or a functional group that enables the compound ofFormula (I) to link with a cell-binding agent, or a functional groupthat enables the compound of Formula (I) to link with a linker attachedon a cell-binding agent, the function group being selected from thegroup consisting of a thiol, an amine, a hydrazine, an alkoxylamino, adisulfide substituent, a maleimido, a haloacetyl group, a carboxy acid,an N-hydroxy succinimide ester, a ketone, an ester, an aldehyde, analkynyl, an alkenyl, and a protected thiol or disulfide group: SAr, SSR₁or SSAr, wherein Ar is an aromatic group or hetero aromatic group, thecell-binding agent being selected from the group consisting ofantibodies and fragments each containing at least one binding site,lymphokines, hormones, growth factors, nutrient-transport molecules andvitamins.
 2. The compound according to claim 1 having the followingFormula (Ia) (Ib), or (Ic):

or a pharmaceutically acceptable salt, hydrate, hydrated salt, orpolymorphic crystalline structure thereof, or an optical isomer,racemate, diastereomer or enantiomer thereof, wherein R₁, R₂, R₄, R₅,R₇, R₈, R₉, R₁₀, L and Q are defined the same as in claim
 1. 3. Thecompound according to claim 1 having the following Formula (Id):

or a pharmaceutically acceptable salt, hydrate, hydrated salt, orpolymorphic crystalline structure thereof, or an optical isomer,racemate, diastereomer or enantiomer thereof; wherein R₁, R₂, R-4, R₅,R₁₀, L and Q are defined the same as in claim
 1. 4. The compoundaccording to claim 1 having the following Formula (Ia-1), (Ia-2),(Ia-3), (Ia-4), (Ia-5), (Ia-6), (Ia-7), (Ia-8), (Ia-9), (Ia-10),(Ia-11), (Ia-12), (Ia-13), (Ia-14), (Ia-15), (Ia-16), (Ia-17), (Ia-18),(Ia-19), (Ia-20), (Ia-21), (Ia-22), (1a-23), (Ia-24), (Ib-1), (Ib-2),(Ib-3), (Ib-4), (Ib-5), (Ib-6), (Ib-7), (Ic-1), (Ic-2), (Ic-3), (Ic-4),(Ic-5), or (Ic-6):

or a pharmaceutically acceptable salt, hydrate, hydrated salt, orpolymorphic crystalline structure thereof, or an optical isomer,racemate, diastereomer or enantiomers thereof; wherein R₁₀, L and Q aredefined the same as in claim
 1. 5. The compound according to claim 1having the following Formula (Id-1), (Id-2), (Id-3), (Id-4), (Id-5),(Id-6), (Id-7), (Id-8), (Id-9), (Id-10), (Id-11), (Id-12), (Id-13),(Id-14), (Id-15), (Id-16), (Id-17), (Id-18), (Id-19), (Id-20), or(Id-21):

wherein L and Q are defined the same as in claim 1; R₁₄ is H, PO₃ ²⁻,SO₃ ⁻, R₁₂, —COR₁₂, —COCH₃, —COOR₁₂, —CONR₁₂R₁₂′, —C(═O)R₁₂NH(Aa)_(t),wherein Aa is an amino acid or a polypeptide, t represents 0-100;—CSNHR₁₂ (thiocarbamate); —SOR₁₂ (sulfoxide); —SO₂R₁₂ (sulfone); —SO₃ ⁻,HSO₃, HSO₂, or a salt of H₅O₃ ⁻, SO₃ ²⁻ or —HSO₂ ⁻ (sulphite);P(O)(OM₁)(OM₂), CH₂OP(O)(OM₁)(OM₂), SO₃M₁; or glycoside (glucoside,galactoside, mannoside, glucuronoside, alloside, fructoside; M₁ and M₂are independently H, Na, K, Ca, Mg, NH₄, or NR₁′R₂′R₃′; R₁′, R₂′ and R₃′are independently H, or C₁˜C₈ alkyl.
 6. The compound according to claim1 having the following Formula (I-1), (I-2), (I-3), (I-4), (I-5), (I-6),(I-7), (I-8), (I-9), (I-10), (I-11), (I-12), (I-13), (I-14), (I-15),(I-16), (I-17), (I-18), (I-19), (I-20), (I-21), (I-22), (I-23), (I-24),(I-25), (I-26), (I-27), (I-28), (I-29), (I-30), (I-31), (I-32), (I-33),(I-34), (I-35), (I-36), (I-37), (I-38), (I-39), (I-40), (I-41), (I-42),(I-43), (I-44), (I-45), (I-46), (I-47), (I-48), (I-49), (I-50), (I-51),(I-52), (I-53), (I-54), (I-55), (I-56), (I-57), (I-58), (I-69), (I-60),(I-61), (I-62), (I-63), (I-64), (I-65), (I-66), (I-67), (I-68), (I-69),(I-70), (I-71), (I-72), (I-73), (I-74), (I-75), (I-76), (I-77), (I-78),(I-79), (I-80), (I-81), (I-82), (I-83), (I-84), (I-85), (I-86), (I-87),(I-88), (I-89), (I-90), (I-91), (I-92), (I-93), (I-94), (I-95), (I-95)or (I-95):

or a pharmaceutically acceptable salt, hydrate, hydrated salt, orpolymorphic crystalline structure thereof, or an optical isomer,racemate, diastereomer or enantiomer thereof, wherein Aa, r, n, p, q andQ are defined the same as in the claim 1; PEG is polyethylene glycolwith the formula of —(OCH₂CH₂)_(r).
 7. A method for preparing thecompound according to claim 1, comprising sequentially (i) aromaticnitration of an indole unit, (ii) reduction of a nitro group on abenzene ring of the indole unit to an amine, and (iii) condensation ofthe produced amine compound with a linker having a reactive or areactable carboxylic group to form an amide linkage as illustratedbelow:

wherein R₁₀, L and Q are defined the same as in claim 1, wherein Lv isleaving group selected from the group consisting of OH, halogen, NHS(N-hydroxyl succinimide), nitrophenol, pentalfluorophenol, and anintermediate generated from peptide coupling and from Mitsunobureaction.
 8. The compound according to claim 1, wherein the linker L isselected from the group consisting of: R₁₂, OR₁₂, OR₁₂O, NHR₁₂, NHR₁₂NH,NR₁₁R₁₂, SR₁₂S, OR₁₂NH, OR₁₂Ar, NHR₁₂Ar, NR₁₁R₁₂NR₁₂′R₁₂″,—(CR₁₁R₁₂)_(p)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)(Aa)_(r)(OCH₂CH₂)_(t)—,-(Aa)_(r)(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(n)(OCH₂CH₂)_(t)(Aa)_(r),—(CR₁₁R₁₂)_(p)(CH═CH)(CR₁₂′R₁₂″)_(q)(Aa)_(r)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(NR₁₂′CO)(Aa)_(r)(CR₁₂′R₁₂′—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(Aa)_(t)(NHCO)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(r),(CR₁₁R₁₂)_(p)(OCO)(Aa)_(r)-(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CO)-(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(NR₁₁CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)—(OCO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)(CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—(CR₁₁R₁₂)_(p)-phenyl-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)—(CR₁₁R₁₂)_(p)-furyl-CO-(Aa)_(t)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-oxazolyl-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-thiazolyl-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-thienyl-CO(CR₁₂′R₁₂″)_(q),—(CR₁₁R₁₂)_(p)-imidazolyl-CO—(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)-morpholino-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—(CR11R₁₂)_(p)-piperazino-CO(Aa)_(r)-(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)—N-methylpiperazin-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—(CR₁₁R₁₂)_(p)(Aa)_(r)-phenyl-, —(CR₁₁R₁₂)_(p)-(Aa)_(r)-furyl-,—(CR₁₁R₁₂)_(p)-oxazolyl(Aa)_(r)-, —(CR₁₁R₁₂)_(p)-thiazolyl-(Aa)_(r),—(CR₁₁R₁₂)_(p)-thienyl-(Aa)_(r)-, —(CR₁₁R₁₂)_(p)-imidazolyl(Aa)_(r),—(CR₁₁R₁₂)_(p)-morpholino-(Aa)_(r)-, —(CR₁₁R₁₂)_(p)-piperazino-(Aa)_(r),—(CR₁₁R₁₂)_(p)—N-methylpiperazino-(Aa)_(r)-,—K(CR₁₁R₁₂)_(p)-(Aa)_(r)(CR₁₂′R₁₂″)_(q) (OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(CR₁₁′R₁₂″)_(q)(Aa)_(r)(OCH₂CH₂)_(t)—,—K(Aa)_(r)(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)—(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(r)(Aa)_(t),—K(CR₁₁R₁₂)_(p)(CR₇═CR₈)(CR₁₂′R₁₂″)_(q)-(Aa)_(r)(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(NR₇CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(Aa)_(t)(NR₇—CO)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(OCO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)p(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t)—,—K(CR₁₁R₁₂)_(p)(CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(—CR₁₁R₁₂)_(p)(NR₁₁CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(OCO)(Aa)_(r)(CR₁₁′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(OCNR₇)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(t),—K(CR₁₁R₁₂)_(p)(CO)(Aa)_(r)(CR₁₂′R₁₂″)_(q)(OCH₂CH₂)_(r)Q,—K(CR₁₁R₁₂)_(p)-phenyl-CO-(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-furyl-CO(Aa)_(t)-(CR₁₂′R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)-oxazolyl-CO(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-thiazolyl-CO(Aa)_(t)-(CR₁₂′R₁₂′)_(q),—K(CR₁₁R₁₂)_(p)-thienyl-CO(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-thiazolyl-CO—(CR₁₂′R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)-morpholino-CO(Aa)_(t)(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)-piperazino-CO-(Aa)_(r)(CR₁₂′R₁₂″)_(q)—,—K(CR₁₁R₁₂)_(p)—N-methylpiperazin-CO(Aa)_(r)-(CR₁₁R₁₂″)_(q),—K(CR₁₁R₁₂)_(p)-(Aa)_(r)-phenyl-, —K(C₁₁R₁₂)_(m)-(Aa)_(r)-furyl-,—K(C₁₁R₁₂)_(p)-oxazolyl(Aa)_(r), —K(CR₁₁R₁₂)_(m)-thiazolyl-(Aa)_(r)-,—K(CR₁₁R₁₂)_(p)-thienyl-(Aa)_(r), —K(CR₁₁R₁₂)_(p)-imidazolyl(Aa)_(r),—K((CR₁₁R₁₂)_(m)-morpholino-(Aa)_(r),—K(CR₁₁R₁₂)_(p)-piperazino-(Aa)_(t)-, and—K(CR₁₁R₁₂)_(m)N-methylpiperazino-(Aa)_(r), wherein K is NR₁₂, O, S, Se,B, or C₃˜C₁₀ of Ar or heterocyclic; wherein Aa, r, n, p, q, t, R₇, R₁₁,R₁₂, R₁₂′, R₁₂″ are as defined in claim
 1. 9. The compound according toclaim 1 having the following formula (II-1)-(II-91):

wherein Aa, L, m, n, p, Q, r, R₁, and R₂, are described the same as inclaim 1; CBA is a cell-binding agent.
 10. The compound of claim 1,wherein the cell binding agent is a full-length antibody (polyclonal andmonoclonal antibody); a single chain antibody; a diabody, a triabody, afragments of antibody (Fab, Fab′, F(ab′)₂, Fv, a fragment produced by aFab expression library, an anti-idiotypic (anti-Id) antibody, CDR's, andan epitope-binding fragment of any of the above whichimmuno-specifically bind to cancer cell antigens, viral antigens ormicrobial antigens; interferon (type I, II, III); a peptide; alymphokine IL-2, IL-3, IL-4, IL-6, GM-CSF, interferon-gamma (IFN-γ); ahormone, insulin, TRH (thyrotropin releasing hormone), MSH(melanocyte-stimulating hormone), a steroid hormone, androgens,estrogens, melanocyte-stimulating hormone (MSH); a growth factor and acolony-stimulating factor, an epidermal growth factors (EGF), agranulocyte-macrophage colony-stimulating factor (GM-CSF), atransforming growth factors (TGF), TGFα, TGFβ; a insulin and insulinlike growth factor (IGF-I, IGF-II) G-CSF, M-CSF and GM-CSF; a vacciniagrowth factor (VGF); a fibroblast growth factor (FGFs); a smallermolecular weight protein, poly-peptide, peptides and peptide hormones,bombesin, gastrin, gastrin-releasing peptide; a platelet-derived growthfactor; an interleukin and a cytokine, interleukin-2 (IL-2),interleukin-6 (IL-6), a leukemia inhibitory factor, agranulocyte-macrophage colony-stimulating factor (GM-CSF); a vitamin,folate; an apoprotein and a lycoproteins; transferrin; a sugar-bindingprotein or a lipoproteins, a lectin; a cell nutrient-transport molecule(transferrin); and a small molecular inhibitor, prostate-specificmembrane antigen (PSMA) inhibitor and small molecular tyrosine kinaseinhibitors (TKI); peptides, or peptide analogs, proteins includingconjugated proteins that are able to bind targeted cells; a non-peptidesor any other cell binding molecule or substance in a form of bioactivepolymer, bioactive dendrimer, nanoparticle, liposome, or viral capside.11. The compound according to claim 1, wherein L is one or more linkercomponents of 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”or “af”), glycine-glycine, a nature peptides containing up to 6 the sameor different natural amino acids (dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide), p-aminobenzyloxycarbonyl (“PAB”),N-succinimidyl 4-(2-pyridylthio)pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”), ethyleneoxy (—CH₂CH₂O—) as one orup to 100 repeating units (“EO” or “PEO”), or one or more componentsthat are illustrated below:

wherein R₁₀ is defined the same as in claim 1; R₁₅, R₁₆ and R₁₇ areindependently selected from the group consisting of —C₁˜C₈ alkyl oralkylene-, carbocyclo-, —O-(C₁˜C₈ alkyl)-, —NH—(C₁˜C₈ alkyl)-,-arylene-, —C₁˜C₈ alkylene-arylene-, -arylene, —C₁˜C₈alkylene-, —C₁˜C₈alkylene-(C₁˜C₈ carbocyclo)-, —(C₃˜C₇ carbocyclo)-C₁˜C₈ alkylene-,—C₃˜C₈ heterocyclo-, alkylene-(C₃˜C₈ heterocyclo)-, —(C₃˜C₈heterocyclo)-C₁˜C₉ alkylene-, —(CH₂CH₂O)_(k)—, —(CH(CH₃)CH₂O)_(k)—, and—(CH₂CH₂O)_(k)—CH₂—; k is an integer ranging from 1-50; X′″, Y″ and Z′″are independently NH, O or S.
 12. The compound according to claim 1,having any one of the specific conjugation structures of (III-1),(III-2), (III-3), (III-4), (III-5), (III-6), (III-7), (III-8), (III-9),(III-10), (III-11), and (III-12) described in FIGS. 31A-31F wherein “═”represents either a single bond or a double bond; L₁ and L₂ are, thesame or different, independently defined the same as L in claim 1; X₁and X₂, are, the same or different, independently NH, N(R₁), O, S, CH₂,or Ar, wherein R₁ is C₁-C₆ alkyl and Ar is an aromatic or heteroaromaticring; wherein Drug₁ and Drug₂ are the same or different, independently amoiety linked to L other than Q in Formula (I) of claim 1, or one ofDrug₁ and Drug₂ is the moiety linked to L other than Q in Formula (I) ofclaim 1, the other of Drug₁ and Drug₂ is absent, (OCH₂CH₂)_(r)OR₁₀,(OCH₂CH(CH₃))_(p)OR₁₀, NH(CH₂CH₂O)_(p)R₁₀, NH(CH₂CH(CH₃)O)_(p)R₁₀,N[(CH₂CH₂O)_(p)R₁₀][(CH₂CH₂O)_(r)R₅], (OCH₂CH₂)_(p)COOR₁₀, orCH₂CH₂(OCH₂CH₂)_(p)COOR₁₀, wherein p, r, R₁₀ are described the same asin claim 1, when both Drug₂ and L₂ are absent, X₂ is NH₂ or OH.
 13. Thecompound according to claim 1, comprising an IgG antibody linked to oneof the following linkage structures of (IV-1), (IV-2), (IV-3), (IV-4),(IV-5), and (IV-6):

wherein “═” represents either a single bond or a double bond; Drug is amoiety linked to L other than Q in Formula (I) of claim 1;

represents a site on an antibody; L, X, n and R₁₂ are defined the sameas in claim
 1. 14. A process for forming the compound according to claim12 comprising a step of reducing a dithiol bond in an antibody with oneor more reducing agent selected from the group consisting ofdithiothreitol (DTT), dithioerythritol (DTE), L-glutathione (GSH), tris(2-carboxyethyl) phosphine (TCEP), 2-mercaptoethylamine (13-MEA), andbeta mercaptoethanol 2-ME), the reducing agent being optionally loadedor covalently bonded to a solid polymer or a solid particle, wherein thepolymer or the particle is selected from the group consisting ofpolyethene, polyacrylate, silica, crossed-linked silica(2-mercaptoethyl)silica, (aminoethyl)silica, (aminopropyl)silica),polyethylene terephthalate, polyethylene glycol, polystyrene,poly(isopropyl acrylate), dextrans (Sephadex, cross-linked dextran),isopropylacrylamide butyl methacrylate copolymer, and polysaccharidepolymer (agarose, agar, agaropectin, Sepharose).
 15. The compoundaccording to claim 12, when one of Drug₁ and Drug₂ is a moiety linked toL other than Q in Formula (I), the other one of Drug₁ and Drug₂ isselected from the group consisting of a protein, an antibody (amonoclonal or polyclonal antibody, antibody dimers, antibody multimers,a bispecific or trispecific antibody, a single chain antibody, anantibody fragment that binds to the target cell), a chromophoremolecule, a tubulysin derivative, a maytansinoid, a taxanoid (taxane), aCC-1065 analog, a daunorubicin or doxorubicin compound, a benzodiazepinedimer (dimers of pyrrolobenzodiazepine (PBD), tomaymycin, anthramycin,indolinobenzodiazepines, imidazobenzothiadiazepines, oroxazolidinobenzodiazepines), a calicheamicin, a dolastatin or auristatinderivative (monomethyl auristatin E, MMAE, MMAF, auristatin PYE,auristatin TP, Auristatins 2-AQ, 6-AQ, EB (AEB), EFP (AEFP)), aduocarmycin, a siRNA, and an enzyme.
 16. A pharmaceutical compositioncomprising a therapeutically effective amount of the compound of claim1, and a pharmaceutically acceptable excipient selected from the groupconsisting of one or more components of 0.002%˜1% of polysorbate(polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80),sodium lauryl sulfate, triton X-100; 0.01% 10% of a binder(disaccharides: sucrose, lactose, trehalose or maltose; or sugaralcohols: xylitol, sorbitol or maltitol, or polyethylene glycol), and0.01%˜10% of a pharmaceutical buffering agent (citrate, succinateacetate, phosphate, or borate) at a pH of 4.5-9.5.
 17. A method forinhibiting abnormal cell growth or treating a proliferative disorderincluding cancers, benign or malignant tumors; leukemia and lymphoidmalignancies; neuronal, glial, astrocytal, hypothalamic, glandular,macrophagal, epithelial, stromal, blastocoelic, angiogenic andimmunologic disorders; inflammatory; autoimmune disorders; destructivedisorders; bone disorder; infectious disease; viral disease; fibroticdisease; neurodegenerative disorder; pancreatitis or kidney disease; ina mammal, comprising administering to the mammal a pharmaceuticallyeffective amount of the compound according to claim
 1. 18. Thepharmaceutical composition according to claim 16 further comprising oneor more synergistic drugs of a chemotherapeutic agent, radiationtherapy, immunotherapy agent, autoimmune disorder agent, oranti-infectious agent.
 19. The compound according to claim 1, whereinthe cell binding agent is an antibody, an antibody fragment, a diabody,a tri(a)body, an epidermal growth factor (EGF), a prostate specificmembrane antigen (PSMA) inhibitor, a melanocyte stimulating hormone(MSH), a thyroid stimulating hormone (TSH), a polyclonal antibody, asomatostatin, a folate, a matriptase inhibitor, an estrogen, an estrogenanalogue, a designed ankyrin repeat proteins (DARPins), an androgen, oran androgen analogue.
 20. The compound according to claim 1, wherein Qtargets cells selected from the group consisting of tumor cells; virusinfected cells; microorganism infected cells; parasite infected cells;autoimmune cells; activated cells; myeloid cells; activated T-cells, Bcells, or melanocytes; cells expressing antigen of CD3, CD4, CD5, CD6,CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16,CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40,CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD51,CD52, CD53, CD54, CD55, CD56, CD58, CD59, CD61, CD62E, CD62L, CD62P,CD63, CD66, CD68, CD69, CD70, CD72, CD74, CD79, CD79a, CD79b, CD80,CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD98,CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD125, CD126, CD127,CD133, CD134, CD135, CD137, CD138, CD141, CD142, CD143, CD144, CD147,CD151, CD147, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD174,CD180, CD184, CDw186, CD194, CD195, CD200, CD200a, CD200b, CD209, CD221,CD227, CD235a, CD240, CD262, CD271, CD274, CD276 (B7-H3), CD303, CD304,CD309, CD326, 4-1BB, SAC, 5T4 (Trophoblast glycoprotein, TPBG, 5T4,Wnt-Activated Inhibitory Factor 1 or WAIF1), Adenocarcinoma antigen,AGS-5, AGS-22M6, Activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alphaintergrin, Alpha v beta6, Amino-peptidase N, Amyloid beta, Androgenreceptor, Angiopoietin 2, Angiopoietin 3, Annexin A1, Anthrax toxinprotective antigen, Anti-transferrin receptor, AOC3 (VAP-1), B7-H3,Bacillus anthracis anthrax, BAFF (B-cell activating factor), BCMA,B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125(carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase9), CALLA, CanAg, Canis lupus familiaris IL31, Carbonic anhydrase IX,Cardiac myosin, CCL11(C-C motif chemokine 11), CCR4 (C-C chemokinereceptor type 4, CD194), CCR5, CD3E (epsilon), CEA (Carcinoembryonicantigen), CEACAM3, CEACAM5 (carcino-embryonic antigen), CFD (Factor D),Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), Clumping factorA, cMet, CRIPTO, FCSFIR (Colony stimulating factor 1 receptor, CD115),CSF2 (colony stimulating factor 2, Granulocyte-macrophagecolony-stimulating factor (GM-CSF)), CSP4, CTLA4 (cytotoxicT-lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4(CD184), C—X—C chemokine receptor type 4, cyclic ADP ribose hydrolase,Cyclin B1, CYP1B1, Cytomegalovirus, Cytomegalovirus glycoprotein B,Dabigatran, DLL4 (delta-like-ligand 4), DPP4 (Dipeptidyl-peptidase 4),DR5 (Death receptor 5), E. coli shiga toxin type-1, E. coli shiga toxintype-2, ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR,EGFRII, EGFRvIII, Endoglin (CD105), Endothelin B receptor, Endotoxin,EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2(Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 ETS fusiongene), Escherichia coli, ETV6-AML, FAP (Fibroblast activation proteinalpha), FCGR1, alpha-Fetoprotein, Fibrin II, beta chain, Fibronectinextra domain-B, FOLR (folate receptor), Folate receptor alpha, Folatehydrolase, Fos-related antigen 1F protein of respiratory syncytialvirus, Frizzled receptor, Fucosyl GM1, GD2 ganglioside, G-28 (a cellsurface antigen glyvolipid), GD3 idiotype, GloboH, Glypican 3,N-glycolylneuraminic acid, GM3, GMCSF receptor a-chain, Growthdifferentiation factor 8, GP100, GPNMB (Trans-membrane glycoproteinNMB), GUCY2C (Guanylate cyclase 2C, guanylyl cyclase C(GC-C), intestinalGuanylate cyclase, Guanylate cyclase-C receptor, Heat-stable enterotoxinreceptor (hSTAR)), Heat shock proteins, Hemagglutinin, Hepatitis Bsurface antigen, Hepatitis B virus, HER1 (human epidermal growth factorreceptor 1), HER2, HER2/neu, HER3 (ERBB-3), IgG4, HGF/SF (Hepatocytegrowth factor/scatter factor), HHGFR, HIV-1, Histone complex, HLA-DR(human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, Human chorionicgonadotropin, HNGF, Human scatter factor receptor kinase, HPV E6/E7,Hsp90, hTERT, ICAM-1 (Intercellular Adhesion Molecule 1), Idiotype,IGFIR (IGF-1, insulin-like growth factor 1 receptor), IGHE, IFN-γ,Influenza hemagglutinin, IgE, IgE Fc region, IGHE, IL-1, IL-2 receptor(interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22,IL-23, IL31RA, ILGF2 (Insulin-like growth factor 2), Integrins (α4,α_(IIb)β₃, αvβ₃, α₄β₇, α₅β₁, α₆β₄, α₇β₇, α₁₁β₃, α₅β₅, αvβ₅), Interferongamma-induced protein, ITGA2, ITGB2, KIR2D, Kappa Ig, LCK, Le, Legumain,Lewis-Y antigen, LFA-1 (Lymphocyte function-associated antigen 1,CD11a), LHRH, LINGO-1, Lipoteichoic acid, LIV1A, LMP2, LTA, MAD-CT-1,MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MARTI,MCP-1, MW (Macrophage migration inhibitory factor, orglycosylation-inhibiting factor (GIF)), MS4A1 (membrane-spanning4-domains subfamily A member 1), MSLN (mesothelin), MUC1 (Mucin 1, cellsurface associated (MUC1) or polymorphic epithelial mucin (PEM)),MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1),MelanA/MART1, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domains subfamilyA), MYCN, Myelin-associated glycoprotein, Myostatin, NA17, NARP-1,NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neuralapoptosis-regulated proteinase 1, NOGO-A, Notch receptor, Nucleolin, Neuoncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (Oxidized low-densitylipoprotein), OY-TES1, P21, p53 nonmutant, P97, Page4, PAP, Paratope ofanti-(N-glycolylneuraminic acid), PAX3, PAX5, PCSK9, PDCD1 (PD-1,Programmed cell death protein 1, CD279), PDGF-Rα (Alpha-typeplatelet-derived growth factor receptor), PDGFR-β, PDL-1, PLAC1,PLAP-like testicular alkaline phosphatase, Platelet-derived growthfactor receptor beta, Phosphate-sodium co-transporter, PMEL 17,Polysialic acid, Proteinase3 (PRI), Prostatic carcinoma, PS(Phosphatidylserine), Prostatic carcinoma cells, Pseudomonas aeruginosa,PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1(RhPI), CD240), Rhesus factor, RANKL, RhoC, Ras mutant, RGS5, ROBO4,Respiratory syncytial virus, RON, ROR1, Sarcoma translocationbreakpoints, SART3, Sclerostin, SLAMF7 (SLAM family member 7), SelectinP, SDCI (Syndecan 1), sLe(a), Somatomedin C, SIP(Sphingosine-1-phosphate), Somatostatin, Sperm protein 17, SSX2, STEAP1(six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn,TAG-72 (tumor associated glycoprotein 72), Survivin, T-cell receptor, Tcell transmembrane protein, TEM1 (Tumor endothelial marker 1), TENB2,Tenascin C (TN-C), TGF-α, TGF-β (Transforming growth factor beta),TGF-β1, TGF-β2 (Transforming growth factor-beta 2), Tie (CD202b), Tie2,TIM-1 (CDX-014), Tn, TNF-α, TNFRSF8, TNFRSF10B (tumor necrosis factorreceptor superfamily member 10B), TNFRSF-13B (tumor necrosis factorreceptor superfamily member 13B), TPBG (trophoblast glycoprotein),TRAIL-R1 (Tumor necrosis apoprosis Inducing ligand Receptor 1), TRAILR2(Death receptor 5 (DR5)), tumor-associated calcium signal transducer 2,tumor specific glycosylation of MUC1, TWEAK receptor, TYRP1(glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A,VEGF-2 (CD309), VEGFR-1, VEGFR2, vimentin, WT1, XAGE 1, cells expressingany insulin growth factor receptors, and epidermal growth factorreceptors.
 21. A method for treatment of a cancer, autoimmune disorder,infectious disease or viral disease in vitro, in vivo, or ex vivo,comprising utilizing the pharmaceutical composition according to claim16.
 22. The compound according to claim 1, wherein the self-immolativelinker component has one of the following structures:

wherein the (*) atom is a point of attachment of additional spacer orreleasable linker unit, or a cytotoxic agent, or the cell binding agent(CBA); X¹, Y¹, Z² and Z³ are independently NH, O, or S; Z¹ isindependently H, NH, O or S, v is 0 or 1; Q¹ is independently H, OH,C₁˜C₆ alkyl, (OCH₂CH₂)_(n)F, Cl, Br, I, OR₁₂, SR₁₂, NR₁₂R₁₂′, N═NR₁₂,N═R₁₂, NR₁₂R₁₂′, NO₂, SOR₁₂R₁₂′, SO₂R₁₂, SO₃R₁₂, OSO₃R₁₂, PR₁₂R₁₂′,POR₁₂R₁₂′, PO₂R₁₂R₁₂′, OPO(OR₁₂)(OR₁₂′), or OCH₂PO(OR₁₂(OR₁₂′), whereinR₁₂ and R₁₂′ are as defined the same as in claim
 1. 23. The compoundaccording to claim 22, wherein R₁₂ and R₁₂′ are independently H, C₁˜C₈alkyl; C₂˜C₈ alkenyl, alkynyl, heteroalkyl; C₃˜C₈ aryl, heterocyclic,carbocyclic, cycloalkyl, heterocycloalkyl, heteroaralkyl, alkylcarbonyl;or a pharmaceutical cation salt thereof.
 24. The compound according toclaim 1, wherein Q is one of the following formulas:

wherein D is H, —NO₂, SO₃ ⁻, CN, or F; Aa, r, p, q, m, and n aredescribed the same as in claim 1; w and w′ are 0 or 1 independently;R₁′, R₂′, R₃′, and R₄′ are independently H, CH₃, C₂H₅, C₃H₇, CH₂OH, orCH₂CH₂OH.
 25. The compound according to claim 6, wherein Q is H, C₁˜C₈of alkyl, alkenyl, alkynyl, aryl, cyclic, cyclohetero, haloalkyl,alkoxy, haloalkoxy alkylamino; halogen; —NO₂; —CN; —SH; —SSCH₃; —SSAc;—SSAr; —SS-Pyridine; —SS-Ar(-NO₂); —S-cell binding agent; or a functiongroup of NHS ester, pentafluorophenyl ester; alkyloxyamine; aldehyde;ketone; carboxyl acid; hydrazine; amine; or thiolactone; or is linkedthe cell binding agent via Stretcher units (Ww) or via Spacer units(Tt), wherein W, w, T, and t are defined the same as in claim
 6. 26. Thecompound according to claim 12, wherein the IgG antibody is IgG1, IgG2,IgG3 or IgG4 antibody.
 27. The compound according to claim 15, whereinthe other one of Drug₁ and Drug₂ is one of structures illustrated below:V-1, an antibody,

-V-37, a siRNA, -V-38, an enzyme or protein linked from N-terminal,-V-39, an enzyme or protein linked from C-terminal, wherein R₁₀ aredescribed the same as in claim 15, and

is a site of link to either linker L₁ or linker L₂.